Hepatocyte growth factor receptor antagonists and uses thereof

ABSTRACT

Hepatocyte growth factor (HGF) receptor antagonists are provided. The HGF receptor antagonists include HGF receptor antibodies and fragments thereof. The HGF receptor antagonists can be employed to block binding of HGF to HGF receptors or substantially inhibit HGF receptor activation. The HGF receptor antagonists may be included in pharmaceutical compositions, articles of manufacture, or kits. Methods of treating cancer using the HGF receptor antagonists are also provided.

RELATED APPLICATIONS

This application is a continuation application claiming priority toapplication Ser. No. 13/012,632, filed Jan. 24, 2011, which is acontinuation of application Ser. No. 12/365,495, filed Feb. 4, 2009,which is a continuation of application Ser. No. 11/591,436, filed Oct.31, 2006, which is a continuation of Ser. No. 10/232,408, filed Sep. 3,2002, which is a continuation of Ser. No. 09/669,971 filed Sep. 26,2000, now U.S. Pat. No. 6,468,529, which is a continuation of Ser. No.08/952,235, filed Feb. 17, 1998, now U.S. Pat. No. 6,207,152, which is a371 of PCT/US96/08094 filed May 31, 1996, and a continuation-in-part of08/460,368 filed Jun. 2, 1995, now U.S. Pat. No. 5,686,292, the entiredisclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This application relates to hepatocyte growth factor receptorantagonists. The application also relates to the use of the antagonistsin therapy or diagnosis of particular pathological conditions inmammals, including cancer.

BACKGROUND OF THE INVENTION

Hepatocyte growth factor (“HGF”) functions as a growth factor forparticular tissues and cell types. HGF was identified initially as amitogen for hepatocytes [Michalopoulos et al., Cancer Res., 44:4414-4419(1984); Russel et al., J. Cell. Physiol., 119:183-192 (1984); Nakamuraet al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984)]. Nakamura etal., supra, reported the purification of HGF from the serum of partiallyhepatectomized rats. Subsequently, HGF was purified from rat platelets,and its subunit structure was determined [Nakamura et al., Proc. Natl.Acad. Sci. USA, 83:6489-6493 (1986); Nakamura et al., FEBS Letters,224:311-316 (1987)]. The purification of human HGF (“huHGF”) from humanplasma was first described by Gohda et al., J. Clin. Invest., 81:414-419(1988).

Both rat HGF and huHGF have been molecularly cloned, including thecloning and sequencing of a naturally occurring variant lacking 5 aminoacids designated “delta5 HGF” [Miyazawa et al., Biochem. Biophys. Res.Comm., 163:967-973 (1989); Nakamura et al., Nature, 342:440-443 (1989);Seki et al, Biochem. Biophys. Res. Commun., 172:321-327 (1990); Tashiroet al., Proc. Natl. Acad. Sci. USA, 87:3200-3204 (1990); Okajima et al.,Eur. J. Biochem., 193:375-381 (1990)].

The mature form of huHGF, corresponding to the major form purified fromhuman serum, is a disulfide linked heterodimer derived by proteolyticcleavage of the human pro-hormone between amino acids R494 and V495.This cleavage process generates a molecule composed of an α-subunit of440 amino acids (M_(r) 69 kDa) and a β-subunit of 234 amino acids (M_(r)34 kDa). The nucleotide sequence of the huHGF cDNA reveals that both theα- and the β-chains are contained in a single open reading frame codingfor a pre-pro precursor protein. In the predicted primary structure ofmature huHGF, an interchain S—S bridge is formed between Cys 487 of theα-chain and Cys 604 in the β-chain [see Nakamura et al., Nature, supra].The N-terminus of the α-chain is preceded by 54 amino acids, startingwith a methionine group. This segment includes a characteristichydrophobic leader (signal) sequence of 31 residues and the prosequence.The α-chain starts at amino acid (aa) 55, and contains four kringledomains. The kringle 1 domain extends from about aa 128 to about aa 206,the kringle 2 domain is between about aa 211 and about aa 288, thekringle 3 domain is defined as extending from about aa 303 to about aa383, and the kringle 4 domain extends from about aa 391 to about aa 464of the α-chain.

The definition of the various kringle domains is based on their homologywith kringle-like domains of other proteins (such as prothrombin andplasminogen), therefore, the above limits are only approximate. To date,the function of these kringles has not been determined. The β-chain ofhuHGF shows high homology to the catalytic domain of serine proteases(38% homology to the plasminogen serine protease domain). However, twoof the three residues which form the catalytic triad of serine proteasesare not conserved in huHGF. Therefore, despite its serine protease-likedomain, huHGF appears to have no proteolytic activity, and the preciserole of the β-chain remains unknown. HGF contains four putativeglycosylation sites, which are located at positions 294 and 402 of theα-chain and at positions 566 and 653 of the β-chain.

Comparisons of the amino acid sequence of rat HGF with that of huHGFhave revealed that the two sequences are highly conserved and have thesame characteristic structural features. The length of the four kringledomains in rat HGF is exactly the same as in huHGF. Furthermore, thecysteine residues are located in exactly the same positions, anindication of similar three-dimensional structures [Okajima et al.,supra; Tashiro et al., supra].

In a portion of cDNA isolated from human leukocytes, in-frame deletionof 15 base pairs was observed. Transient expression of the cDNA sequencein COS-1 cells revealed that the encoded HGF molecule (delta5 HGF)lacking 5 amino acids in the kringle 1 domain was fully functional [Sekiet al., supra].

A naturally occurring huHGF variant has been identified whichcorresponds to an alternative spliced form of the huHGF transcriptcontaining the coding sequences for the N-terminal finger and first twokringle domains of mature huHGF [Chan et al., Science, 254:1382-1385(1991); Miyazawa et al., Eur. J. Biochem., 197:15-22 (1991)]. Thisvariant, designated HGF/NK2, has been proposed by some investigators tobe a competitive antagonist of mature huHGF. Hartmann et al. havereported, however, that HGF/NK2 may retain the ability to cause MDCKcells to scatter [Hartmann et al., Proc. Natl. Acad. Sci.,89:11574-11578 (1992)].

Another HGF variant, designated HGF/NK1, has also been reported to actas a competitive antagonist of HGF [Lokker et al., J. Biol. Chem.,268:17145-17150 (1993); Lokker et al., EMBO J., 11:2503-2510 (1992)].That HGF/NK1 molecule, containing the N-terminal hairpin and the firstkringle domain, was found to block binding of HGF to the HGF receptor onA549 human lung carcinoma cells. It was also found, however, thatcertain concentrations of the HGF/NK1 induced a detectable increase inreceptor tyrosine phosphorylation in the A549 cells, suggesting someagonistic activity. Accordingly, it is believed that the agonist orantagonist action of HGF/NK1 may be dependent upon cell type.

HGF and HGF variants are described further in U.S. Pat. Nos. 5,227,158,5,316,921, and 5,328,837.

A high affinity receptor for HGF has been identified as the product ofthe c-Met proto-oncogene [Bottaro et al., Science, 251:802-804 (1991);Naldini et al., Oncogene, 6:501-504 (1991); WO 92/13097 published Aug.6, 1992; WO 93/15754 published Aug. 19, 1993]. This receptor is usuallyreferred to as “c-Met” or “p190^(MET)” and typically comprises, in itsnative form, a 190-kDa heterodimeric (a disulfide-linked 50-kDa α-chainand a 145-kDa β-chain) membrane-spanning tyrosine kinase protein [Parket al., Proc. Natl. Acad. Sci. USA, 84:6379-6383 (1987)]. Severaltruncated forms of the c-Met receptor have also been described [WO92/20792; Prat et al., Mol. Cell. Biol., 11:5954-5962 (1991)].

The binding activity of HGF to c-Met is believed to be conveyed by afunctional domain located in the N-terminal portion of the HGF molecule,including the first two kringles [Matsumoto et al., Biochem. Biophys.Res. Commun., 181:691-699 (1991); Hartmann et al., Proc. Natl. Acad.Sci., 89:11574-11578 (1992); Lokker et al., EMBO J., 11:2503-2510(1992); Lokker and Godowski, J. Biol. Chem., 268:17145-17150 (1991)].The c-Met protein becomes phosphorylated on tyrosine residues of the145-kDa β-subunit upon HGF binding.

Certain antibodies to this HGF receptor have been reported in theliterature. Several such antibodies are described below.

Prat et al., Mol. Cell. Biol., supra, describe several monoclonalantibodies specific for the extracellular domain of the β-chain encodedby the c-Met gene [see also, WO 92/20792]. The monoclonal antibodieswere selected following immunization of Balb/c mice with whole livingGTL-16 cells (human gastric carcinoma cell line) overexpressing the Metprotein. The spleen cells obtained from the immunized mice were fusedwith Ag8.653 myeloma cells, and hybrid supernatants were screened forbinding to GTL-16 cells. Four monoclonal antibodies, referred to asDL-21, DN-30, DN-31 and DO-24, were selected.

Prat et al., Int. J. Cancer, 49:323-328 (1991) describe using anti-c-Metmonoclonal antibody DO-24 for detecting distribution of the c-Metprotein in human normal and neoplastic tissues [see, also, Yamada etal., Brain Research, 637:308-312 (1994)]. The murine monoclonal antibodyDO-24 was reported to be an IgG2a isotype antibody.

Crepaldi et al., J. Cell Biol., 125: 313-320 (1994) report usingmonoclonal antibodies DO-24 and DN-30 [described in Prat et al., Mol.Cell. Biol., supra] and monoclonal antibody DQ-13 to identifysubcellular distribution of HGF receptors in epithelial tissues and inMDCK cell monolayers. According to Crepaldi et al., monoclonal antibodyDQ-13 was raised against a peptide corresponding to nineteenCOOH-terminal amino acids (from Ser¹³⁷² to) Ser¹³⁹⁰) of the human c-Metsequence.

A monoclonal antibody specific for the cytoplasmic domain of human c-Methas also been described [Bottaro et al., supra].

Several of the monoclonal antibodies referenced above are commerciallyavailable from Upstate Biotechnology Incorporated, Lake Placid, N.Y.Monoclonal antibodies DO-24 and DL-21, specific for the extracellularepitope of c-Met, are available from Upstate Biotechnology Incorporated.Monoclonal antibody DQ-13, specific for the intracellular epitope ofc-Met, is also available from Upstate Biotechnology Incorporated.

In addition to binding c-Met, it is recognized that HGF binds to someheparin and heparan sulfate proteoglycans which are present on cellsurfaces or in extracellular matrices [Rouslahti et al., Cell,64:867-869 (1991); Lyon et al., J. Biol. Chem., 269:11216-11223 (1994)].Heparan sulfate is a glycosaminoglycan similar in composition andstructure to heparin and is found on many mammalian cell surfaces.Various hypotheses have been proposed to explain the role of heparin andheparan sulfate proteoglycans (“HSPGs”) in the regulation of certaingrowth factor activity. For example, it has been hypothesized that uponbinding heparin or HSPGs, certain growth factors may have a morefavorable conformation for binding to their respective high affinityreceptors [Lindahl et al., Annual Rev. Biochem., 47:385-417 (1995)];that HSPGs may serve as docking sites for certain growth factorsfacilitating the presentation of ligand to its high affinity receptor[Yayon et al., Cell, 64:841-848 (1991); Moscatelli et al., J. Biol.Chem., 267:25803-25809 (1992); Nugent et al., Biochemistry, 31:8876-8883(1992)]; and that HSPGs may promote ligand dimerization facilitatingreceptor activation [Ornitz et al., Mol. Cell. Biol., 12:240-247 (1992);Spivak-Kroizman et al., Cell, 79:1015-1024 (1994)]. It has further beenpostulated that certain growth factors are more stable or resistant toproteolytic activity [Damon et al., J. Cell. Physiol., 138:221-226(1989); Mueller et al., J. Cell. Physiol., 140:439-448 (1989); Rosengartet al., Biochem. Biophys. Res. Commun., 152:432-440 (1998)] anddenaturation [Copeland et al., Arch. Biochem. Biophys., 289:53-61(1994)] when bound to heparin. Coincubation of HGF with soluble heparinand other heparin-like molecules has been reported to promotedimerization/oligomerization of HGF and to potentiate HGF mitogenicactivity. [see, e.g., WO 94/09969 published Mar. 16, 1995; Zioncheck etal., J. Biol. Chem., 270:16871-16878 (1995)].

Mizuno et al. describe some experiments which attempted to locateheparin-binding sites within the HGF molecule [Mizuno et al., J. Biol.Chem., 269:1131-1136 (1994)]. Mizuno et al. constructed variouslydeleted mutant HGFs [d-K1 (deletion of first kringle domain); d-K2(deletion of second kringle domain); d-K3 (deletion of third kringledomain); d-K4 (deletion of fourth kringle domain); d-beta (deletion ofbeta chain); d-H (deletion of N-terminal hairpin loop); and HK1K2(consisting of N-terminal hairpin loop and the first and second kringledomains)] and examined their respective binding to an immobilizedheparin column. The reference reports that the d-H and d-K2 mutantsexhibited decreased binding to heparin affinity columns, while thenative HGF and the other constructed HGF mutants tightly bound to theheparin columns.

Various biological activities have been described for HGF and its c-Metreceptor [see, generally, Chan et al., Hepatocyte Growth Factor-ScatterFactor (HGF-SF) and the C-Met Receptor, Goldberg and Rosen, eds.,Birkhauser Verlag-Base1 (1993), pp. 67-79]. It has been observed thatlevels of HGF increase in the plasma of patients with hepatic failure[Gohda et al., supra] and in the plasma [Lindroos et al., Hepatol.,13:734-750 (1991)] or serum [Asami et al., J. Biochem., 109:8-13 (1991)]of animals with experimentally induced liver damage. The kinetics ofthis response are usually rapid, and precedes the first round of DNAsynthesis during liver regeneration. HGF has also been shown to be amitogen for certain cell types, including melanocytes, renal tubularcells, keratinocytes, certain endothelial cells and cells of epithelialorigin [Matsumoto et al., Biochem. Biophys. Res. Commun., 176:45-51(1991); Igawa et al., Biochem. Biophys. Res. Commun., 174:831-838(1991); Han et al., Biochem., 30:9768-9780 (1991); Rubin et al., Proc.Natl. Acad. Sci. USA, 88:415-419 (1991)]. Both HGF and the c-Metprotooncogene have been postulated to play a role in microglialreactions to CNS injuries [DiRenzo et al., Oncogene, 8:219-222 (1993)].

HGF can also act as a “scatter factor”, an activity that promotes thedissociation of epithelial and vascular endothelial cells in vitro[Stoker et al., Nature, 327:239-242 (1987); Weidner et al., J. CellBiol., 111:2097-2108 (1990); Naldini et al., EMBO J., 10:2867-2878(1991); Giordano et al., Proc. Natl. Acad. Sci. USA, 90:649-653 (1993)].Moreover, HGF has recently been described as an epithelial morphogen[Montesano et al., Cell, 67:901-908 (1991)]. Therefore, HGF has beenpostulated to be important in tumor invasion [Comoglio, HepatocyteGrowth Factor-Scatter Factor (HGF-SF) and the C-Met Receptor, Goldbergand Rosen, eds., Birkhauser Verlag-Base1 (1993), pp. 131-165]. Bellusciet al., Oncogene, 9:1091-1099 (1994) report that HGF can promotemotility and invasive properties of NBT-II bladder carcinoma cells.

c-Met RNA has been detected in several murine myeloid progenitor tumorcell lines [Iyer et al., Cell Growth and Differentiation, 1:87-95(1990)]. Further, c-Met is expressed in various human solid tumors [Pratet al., Int. J. Cancer, supra]. Overexpression of the c-Met oncogene hasalso been suggested to play a role in the pathogenesis and progressionof thyroid tumors derived from follicular epithelium [DiRenzo et al.,Oncogene, 7:2549-2553 (1992)]. Chronic c-Met/HGF receptor activation hasalso been observed in certain malignancies [Cooper et al., EMBO J.,5:2623 (1986); Giordano et al., Nature, 339:155 (1989)].

In view of the role of HGF and/or c-Met in potentiating or promotingsuch diseases or pathological conditions, it would be useful to have ameans of substantially reducing or inhibiting one or more of thebiological effects of HGF and c-Met.

SUMMARY OF THE INVENTION

The invention provides HGF receptor antagonists which are capable ofspecifically binding to a HGF receptor. Preferred HGF receptorantagonists are capable of substantially reducing or inhibiting themitogenic, motogenic (migration or scatter) or other biological activityof HGF or HGF receptor activation, and thus are useful in the treatmentof various diseases and pathological conditions such as cancer. In oneembodiment of the invention, the HGF receptor antagonist is an antibody.Preferably, this antagonist is a monoclonal antibody, and morepreferably, is a Fab fragment of a monoclonal antibody.

The invention also provides hybridoma cell lines which produce HGFreceptor antagonist monoclonal antibodies.

The invention also provides HGF receptor antagonists that compriseisolated polypeptide comprising the amino acid sequences of FIG. 1A (SEQID NO:1) and FIG. 1B (SEQ ID NO:2). The polypeptides consisting of theamino acid sequences of FIG. 1A (SEQ ID NO:1) and FIG. 1B (SEQ ID NO:2)correspond to the light chain and heavy chain, respectively, ofmonoclonal antibody 5D5 Fab, described herein.

The invention also provides chimeric molecules comprising HGF receptorantagonist linked or fused to another, heterologous polypeptide orpolymer. An example of such a chimeric molecule comprises a HGF receptorantagonist amino acid sequence linked or fused to an albumin sequence orpolyethylene glycol (“PEG”) sequence.

The invention further provides an isolated nucleic acid moleculeencoding HGF receptor antagonist. In one aspect, the nucleic acidmolecule is RNA or DNA that encodes a HGF receptor antagonist or iscomplementary to a nucleic acid sequence encoding such HGF receptorantagonist, and remains stably bound to it under stringent conditions.In one embodiment, the nucleic acid sequences are selected from:

(a) the nucleic acid sequence of FIG. 1A that codes for residue 1 toresidue 220 (i.e., nucleotides 1 through 660; SEQ ID NO:3), inclusive;

(b) the nucleic acid sequence of FIG. 1B that codes for residue 1 toresidue 230 (i.e., nucleotides 1 through 690; SEQ ID NO:4), inclusive;or

(c) a nucleic acid sequence corresponding to the sequence of (a) or (b)within the scope of degeneracy of the genetic code.

The invention also provides a replicable vector comprising the nucleicacid molecule(s) encoding the HGF receptor antagonist operably linked tocontrol sequence(s) recognized by a host cell transfected or transformedwith the vector. A host cell comprising the vector or the nucleic acidmolecule(s) is also provided. A method of producing HGF receptorantagonist which comprises culturing a host cell comprising the nucleicacid molecule(s) and recovering the protein from the host cell cultureis further provided.

The invention also provides pharmaceutical compositions comprising oneor more HGF receptor antagonists in a pharmaceutically-acceptablecarrier. In one embodiment, the pharmaceutical composition may beincluded in an article of manufacture or kit.

The invention also provides methods of employing HGF receptorantagonists, including methods of inhibiting HGF receptor activation.

The invention further provides methods for treating cancer comprisingadministering to a mammal diagnosed as having cancer an effective amountof a HGF receptor antagonist. The HGF receptor antagonist alone may beadministered to the mammal, or alternatively, may be administered to themammal in combination with other therapeutic agents such as anti-canceragents.

It is believed that the antagonists can be used to block binding of HGFto HGF receptor(s) or substantially prevent HGF receptor activation,thereby treating pathologic conditions associated with binding of HGF toHGF receptor(s) or with the activation of HGF receptor(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the amino acid sequences (and encoding nucleotides)for the light chain (FIG. 1A) and heavy chain (FIG. 1B), respectively,of monoclonal antibody 5D5 Fab.

FIG. 2 is a graph showing the inhibition of HGF binding to c-Met-IgGfusion protein by monoclonal antibody 1A3.3.13.

FIG. 3 is a bar diagram showing the stimulatory effect of monoclonalantibodies 3D6, 6G1, and 1A3.3.13 on human mammary epithelial cells in aproliferation assay.

FIG. 4 is a bar diagram showing the stimulatory effect of monoclonalantibodies 3D6, 05-237 and 05-238 on mink lung cells in a proliferationassay.

FIG. 5 is a bar diagram showing the inhibitory effect of monoclonalantibody 1A3.3.13 Fab fragments on BaF3-hmet.8 cells in a proliferationassay.

FIGS. 6A and 6B are FACS analysis graphs showing binding specificity ofmonoclonal antibody 5D5 to BaF3-hmet.8 cells expressing c-Met.

FIG. 7 is a graph showing the inhibition of HGF binding to c-Met-IgGfusion protein by monoclonal antibody 5D5 and by 5D5 Fab.

FIGS. 8A and 8B are graphs showing the inhibitory effect of 5D5 Fab onBaF3-hmet.8 cells in a proliferation assay.

FIG. 9 is a graph showing the inhibitory effect of 5D5 Fab on a humanbreast carcinoma cell line (MDA-MB-435) which expresses c-Met.

FIGS. 10A and 10B are bar diagrams showing the inhibitory effect of 5D5Fab on c-Met tyrosine phosphorylation.

FIGS. 11A-11C are graphs comparing inhibitory effects of NK1 (FIG. 11A),5D5 Fab (FIG. 11B), and 5D5 Fab and rhuHGF (FIG. 11C) on BaF3-hmet.8cells in a proliferation assay conducted in the presence or absence ofheparin.

FIG. 12 is a restriction map of plasmid p5D5 containing the discistronicoperon for expression of the chimer 5D5 Fab.

FIG. 13 is a graph showing the inhibition of HGF binding to c-Met-IgGfusion protein by recombinant 5D5 Fab.

FIGS. 14A-14D are graphs comparing the inhibitory effect of recombinant5D5 Fab and recombinant anti-VEGF Fab (control Fab) on BaF3-hmet.8 cellsin a proliferation assay conducted in the presence or absence ofheparin.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the terms “hepatocyte growth factor” and “HGF” refer toa growth factor typically having a structure with six domains (finger,Kringle 1, Kringle 2, Kringle 3, Kringle 4 and serine protease domains)and having the property of binding to a HGF receptor, as defined below.The terms “hepatocyte growth factor” and “HGF” include hepatocyte growthfactor from humans (“huHGF”) and any non-human mammalian species, and inparticular rat HGF. The terms as used herein include mature, pre,pre-pro, and pro forms, purified or isolated from a natural source,chemically synthesized or recombinantly produced. Human HGF is encodedby the cDNA sequence published by Miyazawa et al., 1989, supra, orNakamura et al., 1989, supra. The sequences reported by Miyazawa et al.and Nakamura et al. differ in 14 amino acids. The reason for thedifferences is not entirely clear; polymorphism or cloning artifacts areamong the possibilities. Both sequences are specifically encompassed bythe foregoing terms. It will be understood that natural allelicvariations exist and can occur among individuals, as demonstrated by oneor more amino acid differences in the amino acid sequence of eachindividual. The HGF of the invention preferably has at least about 80%sequence identity, more preferably at least about 90% sequence identity,and even more preferably, at least about 95% sequence identity with anative mammalian HGF. The terms “hepatocyte growth factor” and “HGF”specifically include the delta5 huHGF as disclosed by Seki et al.,supra.

The terms “HGF receptor” and “c-Met” when used herein refer to acellular receptor for HGF, which typically includes an extracellulardomain, a transmembrane domain and an intracellular domain, as well asvariants and fragments thereof which retain the ability to bind HGF. Theterms “HGF receptor” and “c-Met” include the polypeptide molecule thatcomprises the full-length, native amino acid sequence encoded by thegene variously known as p190^(MET).

The present definition specifically encompasses soluble forms of HGFreceptor, and HGF receptor from natural sources, synthetically producedin vitro or obtained by genetic manipulation including methods ofrecombinant DNA technology. The HGF receptor variants or fragmentspreferably share at least about 65% sequence identity, and morepreferably at least about 75% sequence identity with any domain of thehuman c-Met amino acid sequence published in Rodrigues et al., Mol.Cell. Biol., 11:2962-2970 (1991); Park et al., Proc. Natl. Acad. Sci.,84:6379-6383 (1987); or Ponzetto et al., Oncogene, 6:553-559 (1991).

The term “5D5 Fab” is used herein to refer to polypeptide comprisingamino acid residues 1 to 220 of the amino acid sequence shown in FIG. 1A(SEQ ID NO:1) and amino acid residues 1 to 230 of the amino acidsequence shown in FIG. 1B (SEQ ID NO:2), as well as biologically activedeletional, insertional, or substitutional variants thereof. In apreferred embodiment, the 5D5 Fab consists of the amino acid sequencesshown in FIGS. 1A and 1B, which correspond to the light chain and heavychain, respectively, of monoclonal antibody 5D5 Fab. In anotherpreferred embodiment, the biologically active variants have at leastabout 80% sequence identity, more preferably at least about 90% sequenceidentity, and even more preferably, at least about 95% sequence identitywith the sequences described above. The definition encompasses 5D5 Fabobtained from an antibody source, such as papain digestion of monoclonalantibody 5D5 described herein, or prepared by recombinant or syntheticmethods, described for instance in Example 13 below.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising HGF receptor antagonist, or a portion thereof,fused to a “tag polypeptide”. The tag polypeptide has enough residues toprovide an epitope against which an antibody can be made, yet is shortenough such that it does not interfere with activity of the antagonist.The tag polypeptide preferably also is fairly unique so that theantibody does not substantially cross-react with other epitopes.Suitable tag polypeptides generally have at least six amino acidresidues and usually between about 8 to about 50 amino acid residues(preferably, between about 10 to about 20 residues).

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo half-life of theIgG molecule.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the HGF receptorantagonist natural environment will not be present. Ordinarily, however,isolated polypeptide will be prepared by at least one purification step.Homogeneity here means less than about 5% contamination with othersource proteins and polypeptides.

An “isolated” HGF receptor antagonist nucleic acid molecule is a nucleicacid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the HGF receptor antagonist nucleic acid. Anisolated HGF receptor antagonist nucleic acid molecule is other than inthe form or setting in which it is found in nature. Isolated HGFreceptor antagonist nucleic acid molecules therefore are distinguishedfrom the HGF receptor antagonist nucleic acid molecule as it exists innatural cells. However, an isolated HGF receptor antagonist nucleic acidmolecule includes HGF receptor antagonist nucleic acid moleculescontained in cells that ordinarily express HGF receptor antagonistwhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-α-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine. The amino acids are identifiedby either the single-letter or three-letter designations:

Asp D aspartic acid Ile I isoleucine Thr T threonine Leu L leucine Ser Sserine Tyr Y tyrosine Glu E glutamic acid Phe F phenylalanine Pro Pproline His H histidine Gly G glycine Lys K lysine Ala A alanine Arg Rarginine Cys C cysteine Trp W tryptophan Val V valine Gln Q glutamineMet M methionine Asn N asparagine

In the Sequence Listing and Figures, certain other single-letter orthree-letter designations are employed to refer to and identify two ormore amino acids at a given position in the amino acid sequence. Forinstance, at amino acid residue 1 in SEQ ID NO:2, the three-letterdesignation “Glx” is employed to identify that at residue 1, the aminoacid may be a glutamine or a glutamic acid residue.

Nucleotide bases referred to in the Sequence Listing and Figures include“A” (adenine), “C” (cytosine), “G” (guanine), “T” (thymine) and “S”(cytosine or guanine).

The term “heparin” is used in a broad sense and refers to aheterogeneous group of sulfated, straight-chain anionicmucopolysaccharides, often referred to as glycosaminoglycans. Althoughothers may be present, the main sugars in heparin are: α-L-iduronic acid2-sulfate, 2-deoxy-2-sulfamino-α-glucose 6-sulfate, β-D-glucuronic acid,2-acetamido-2-deoxy-α-D-glucose, and L-iduronic acid. These andoptionally other sugars are typically joined by glycosidic linkages. Themolecular weight of heparin typically varies from about 6,000 to about20,000 Da depending on the source and the method of molecular weightdetermination. Heparin is a native constituent of various cells andtissues, especially liver and lung, in several mammalian species.

The term “heparin-independent” when used herein describes HGF receptorantagonists which have substantially reduced ability to bind heparin orare unable to bind heparin, or heparin-like glycosaminoglycans,including heparan sulfate and proteoglycans. Determination of whether aHGF receptor antagonist is heparin-independent can be determined by theskilled artisan without undue experimentation. Heparin-independence canbe determined, for example, by assaying the antagonist for HGF blockingactivity in the presence of heparin, as described in the Examples, andobserving the activity of the molecule.

The terms “agonist” and “agonistic” when used herein refer to ordescribe a molecule which is capable of, directly or indirectly,substantially inducing, promoting or enhancing HGF biological activityor HGF receptor activation.

The terms “antagonist” and “antagonistic” when used herein refer to ordescribe a molecule which is capable of, directly or indirectly,substantially counteracting, reducing or inhibiting HGF biologicalactivity or HGF receptor activation.

The term “HGF biological activity” when used herein refers to anymitogenic, motogenic or morphogenic activities of HGF or any activitiesoccurring as a result of HGF binding to a HGF receptor. The term “HGFreceptor activation” refers to HGF receptor dimerization or HGFreceptor-induced tyrosine kinase activity. HGF receptor activation mayoccur as a result of HGF binding to a HGF receptor, but mayalternatively occur independent of any HGF binding to a HGF receptor.HGF biological activity may, for example, be determined in an in vitroor in vivo assay of hepatocyte growth promotion. Adult rat hepatocytesin primary culture have been used to test the effect of HGF onhepatocyte proliferation. Accordingly, the effect of a HGF receptorantagonist can be determined in an assay suitable for testing theability of HGF to induce DNA synthesis of rat hepatocytes in primarycultures. Human hepatocytes can be cultured similarly to the methodsestablished for preparing primary cultures of normal rat hepatocytes.Alternatively, the effect of a HGF receptor antagonist can be determinedin an assay suitable for testing the ability of HGF to induce DNAsynthesis in other types of cells expressing HGF receptor(s), such asmink lung cells or human mammary epithelial cells described in Examples4 and 5. DNA synthesis can, for example, be assayed by measuringincorporation of ³H-thymidine into DNA. The effectiveness of the HGFreceptor antagonist can be determined by its ability to blockproliferation and incorporation of the ³H-thymidine into DNA. The effectof HGF receptor antagonists can also be tested in vivo in animal models.The term “antibody” is used herein in a broad sense and includes intactimmunoglobulin or antibody molecules, polyclonal antibodies,multispecific antibodies (i.e., bispecific antibodies formed from atleast two intact antibodies) and immunoglobulin fragments (such as Fab,F(ab′)₂, or Fv), so long as they exhibit any of the desired antagonisticproperties described herein.

Antibodies are typically proteins or polypeptides which exhibit bindingspecificity to a specific antigen. Native antibodies are usuallyheterotetrameric glycoproteins, composed of two identical light (L)chains and two identical heavy (H) chains. Typically, each light chainis linked to a heavy chain by one covalent disulfide bond, while thenumber of disulfide linkages varies between the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains [Chothia etal., J. Mol. Biol., 186:651-663 (1985); Novotny and Haber, Proc. Natl.Acad. Sci. USA, 82:4592-4596 (1985)]. The light chains of antibodiesfrom any vertebrate species can be assigned to one of two clearlydistinct types, called kappa (K) and lambda (A), based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains, immunoglobulinscan be assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3,and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α,delta, epsilon, y, and p, respectively.

“Antibody fragments” comprise a portion of an intact antibody, generallythe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments,diabodies, single chain antibody molecules, and multispecific antibodiesformed from antibody fragments.

The term “variable” is used herein to describe certain portions of thevariable domains which differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies [see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, NationalInstitutes of Health, Bethesda, Md. (1987)]. The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity [U.S. Pat. No. 4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)].

The terms “cancer” and “cancerous” when used herein refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. Examples of cancer include but are notlimited to, carcinoma, lymphoma, sarcoma, blastoma and leukemia. Moreparticular examples of such cancers include squamous cell carcinoma,lung cancer (small cell and non-small cell), gastrointestinal cancer,liver cancer, kidney cancer, pancreatic cancer, cervical cancer, bladdercancer, hepatoma, breast cancer, colon carcinoma, and head and neckcancer. While the term “cancer” as used herein is not limited to any onespecific form of the disease, it is believed that the methods of theinvention will be particularly effective for cancers which are found tobe accompanied by increased levels of HGF or overexpression oractivation of HGF receptor in the mammal.

The terms “treating,” “treatment,” and “therapy” as used herein refer tocurative therapy, prophylactic therapy, and preventative therapy.

The term “mammal” as used herein refers to any animal classified as amammal, including humans, cows, horses, dogs and cats. In a preferredembodiment of the invention, the mammal is a human.

II. Compositions and Methods of the Invention

In one embodiment of the invention, HGF receptor antagonists areprovided. Non-limiting examples of HGF receptor antagonists includeantibodies, polypeptides, glycoproteins, glycopeptides, glycolipids,polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules,peptidomimetics, pharmacological agents and their metabolites,transcriptional and translation control sequences, and the like.

A. Antibody Compositions

In one embodiment of invention, the HGF receptor antagonists of theinvention comprise HGF receptor antibodies. For instance, the antagonistantibodies may be polyclonal antibodies. Methods of preparing polyclonalantibodies are known to the skilled artisan. Polyclonal antibodies canbe raised in a mammal, for example, by one or more injections of animmunizing agent and, if desired, an adjuvant. Typically, the immunizingagent and/or adjuvant will be injected in the mammal by multiplesubcutaneous or intraperitoneal injections. Preferably, the immunizingagent includes the c-Met polypeptide or a fusion protein thereof. It maybe useful to conjugate the immunizing agent to a protein known to beimmunogenic in the mammal being immunized. Examples of such immunogenicproteins which may be employed include but are not limited to keyholelimpet hemocyanin, serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. An aggregating agent such as alum may also beemployed to enhance the mammal's immune response. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation. The mammal can then be bled, and theserum assayed for HGF receptor antibody titer. If desired, the mammalcan be boosted until the antibody titer increases or plateaus.

The antagonist antibodies of the invention may, alternatively, bemonoclonal antibodies. Antagonist monoclonal antibodies of the inventionmay be prepared using hybridoma methods, such as those described byKohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, amouse or other appropriate host animal, is typically immunized (such asdescribed above) with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

Preferably, the immunizing agent includes the c-Met polypeptide or afusion protein thereof. The immunizing agent may alternatively comprisea fragment or portion of HGF or a HGF receptor having one or more aminoacid residues that participate in the binding of HGF to its receptor. Ina more preferred embodiment, the immunizing agent comprises anextracellular domain of c-Met fused to an IgG sequence, such asdescribed in Example 1. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Rockville, Md. An example of such a murine myeloma cell lineis P3X63AgU.1, described in Example 1 below. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies [Kozbor, J. Immunol.,133:3001-3005 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against a HGFreceptor. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art, and are described further in the Examples below.The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson and Pollard, Anal.Biochem., 107:220-239 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, E. coli, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for a HGF receptor and another antigen-combining site havingspecificity for a different antigen, such as HER2 or CD3.

It is believed, however, that monovalent antibodies capable of bindingto a HGF receptor will be especially useful as HGF receptor antagonists.While not being bound to any particular theory, it is presently believedthat activation of c-Met may proceed by a mechanism wherein huHGFbinding to c-Met induces aggregation or dimerization of the receptorswhich in turn activates intracellular receptor kinase activity. Becausemonovalent antibodies will likely be unable to induce such aggregationor dimerization, the monovalent antibodies should not activate c-Met.Such monovalent antibodies may be directed against the HGF binding siteof the receptor or may otherwise be capable of interfering with HGF, itsfragments or its variants binding to the HGF receptor, such as bysterically hindering HGF, its fragments or its variants access to thereceptor. Alternatively, the monovalent antibodies may be capable ofsterically preventing HGF receptor dimerization.

Methods for preparing monovalent antibodies are well known in the art.For example, one method involves recombinant expression ofimmunoglobulin light chain and modified heavy chain. The heavy chain istruncated generally at any point so as to prevent heavy chaincrosslinking. Alternatively, the relevant cysteine residues may besubstituted with another amino acid residue or are deleted so as toprevent crosslinking. Recombinant expression of Fab light chain andheavy chain is described in further detail below.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion is also described inExamples 6 and 7 below. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields an F(ab′)₂ fragment that has two antigencombining sites and is still capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain theconstant domains of the light chain and the first constant domain (CH₁)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chainCH₁ domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

In a preferred embodiment of the invention, the antagonists comprise Fabfragments of monoclonal antibodies specific for c-Met. In a morepreferred embodiment, the monoclonal antibody Fab fragments have thesame biological characteristics as the monoclonal antibody Fab fragmentsproduced by digesting either of the monoclonal antibodies secreted bythe hybridoma cell lines deposited under American Type CultureCollection Accession Nos. ATCC HB-11894 or ATCC HB-11895. The term“biological characteristics” is used to refer to the in vitro and/or invivo activities of the monoclonal antibody, e.g., ability tosubstantially reduce or inhibit binding of huHGF to c-Met or tosubstantially reduce or inhibit c-Met activation. Accordingly, themonovalent antibody preferably binds to substantially the same epitopeas the 1A3.3.13 antibody or the 5D5.11.6 antibody disclosed herein. Thiscan be determined by conducting assays described herein and in theExamples. For instance, to determine whether a monoclonal antibody hasthe same specificity as the 1A3.3.13 antibody specifically disclosed(i.e., the antibody having the ATCC deposit No. HB-11894) or the5D5.11.6 antibody specifically disclosed (i.e., the antibody having theATCC deposit No. HB-11895), one can use a competitive ELISA bindingassay such as those described in the Examples. In an even more preferredembodiment, the monoclonal antibody Fab fragments areheparin-independent antagonists, as defined herein. In a preferredembodiment of the invention, the monoclonal antibody or fragment thereofwill inhibit the binding of HGF, its fragments or its variants, or themitogenic activity of HGF, its fragments, or its variants at least about50%, preferably, greater than about 80%, and more preferably, greaterthan about 90%, as determined by an in vitro competitive binding assayor proliferation assay, such as described in the Examples below.

In addition to the antagonist antibodies described above, it iscontemplated that chimeric or hybrid antagonist antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

The antagonist antibodies of the invention may also comprise diabodies.The term “diabodies” refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy chain variable domain(V_(H)) connected to a light chain variable domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen binding sites. Diabodies are described in furtherdetail, for example, in EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci., 90:6444-6448 (1993).

The antagonist antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Reichmann etal., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important in order to reduceantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework (FR) for the humanized antibody [Sims et al., J. Immunol.,151:2296 (1993); Chothia and Lesk, J. Mol. Biol., 196:901 (1987)].Another method uses a particular framework derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies [Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol., 151:2623-2632 (1993)].

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding[see, WO 94/04679 published 3 Mar. 1994].

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (J_(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge [see, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno., 7:33 (1993)]. Humanantibodies can also be produced in phage display libraries [Hoogenboomand Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cote et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].

B. Polypeptides and Nucleic Acid Compositions

The present invention also provides HGF receptor antagonists comprisingone or more isolated polypeptides. In one embodiment, the antagonistcomprises residues 1 to 220 of the amino acid sequence shown in FIG. 1A(SEQ ID NO:1) and residues 1 to 230 of the amino acid sequence shown inFIG. 1B (SEQ ID NO:2). Preferably, the antagonist comprises two isolatedpolypeptides which correspond to the light chain and heavy chain,respectively, of an anti-HGF receptor monoclonal antibody Fab.

1. Preparation of HGF Receptor Antagonist

The description below relates primarily to production of the HGFreceptor antagonist by culturing cells transformed or transfected with avector containing the antagonist nucleic acid and recovering thepolypeptide(s) from the cell culture. It is of course, contemplated thatalternative methods, which are well known in the art, may be employed toprepare the HGF receptor antagonist polypeptide.

1. Isolation of DNA Encoding HGF Receptor Antagonist

The DNA encoding the HGF receptor antagonist may be obtained from anycDNA library prepared from tissue believed to possess the antagonistmRNA and to express it at a detectable level. Accordingly, human c-Metantagonist DNA can be conveniently obtained from a cDNA library preparedfrom human tissues. The c-Met antagonist-encoding gene may also beobtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the c-Metreceptor antagonist or oligonucleotides of at least about 20-80 bases)designed to identify the gene of interest or the protein encoded by it.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding the receptor antagonist is to use PCR methodology [Sambrook etal., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1995)].

One method of screening employs selected oligonucleotide sequences toscreen cDNA libraries from various human tissues. The oligonucleotidesequences selected as probes should be of sufficient length andsufficiently unambiguous that false positives are minimized. Theoligonucleotide is preferably labeled such that it can be detected uponhybridization to DNA in the library being screened. Methods of labelingare well known in the art, and include the use of radiolabels like³²P-labeled ATP, biotinylation or enzyme labeling.

Nucleic acid having all the protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the amino acidsequence disclosed herein, and, if necessary, using conventional primerextension procedures as described in Sambrook et al., supra, to detectprecursors and processing intermediates of mRNA that may not have beenreverse-transcribed into cDNA.

Amino acid sequence variants of the antagonist polypeptide can beprepared by introducing appropriate nucleotide changes into its DNA, orby synthesis of the desired antagonist polypeptide. Such variantsrepresent insertions, substitutions, and/or deletions of residues withinor at one or both of the ends of the amino acid sequences shown in FIGS.1A and 1B for the 5D5 Fab. Any combination of insertion, substitution,and/or deletion can be made to arrive at the final construct, providedthat the final construct possesses the desired antagonistic activity asdefined herein. In a preferred embodiment, the variants have at leastabout 80% sequence identity, more preferably, at least about 90%sequence identity, and even more preferably, at least about 95% sequenceidentity with the sequences identified herein for the 5D5 Fab.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Theseinclude oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis.

2. Insertion of Nucleic Acid into A Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the receptorantagonist may be inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector components generally include, but are notlimited to, one or more of the following: a signal sequence, an originof replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, each of which isdescribed below.

(i) Signal Sequence Component

The HGF receptor antagonist may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the antagonist DNA that is inserted intothe vector. The heterologous signal sequence selected preferably is onethat is recognized and processed (i.e., cleaved by a signal peptidase)by the host cell. The signal sequence may be a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders, for example, the herpessimplex glycoprotein D signal.

The DNA for such precursor region is preferably ligated in reading frameto DNA encoding the antagonist.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used because it contains the earlypromoter).

Most expression vectors are “shuttle” vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example, a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of the receptor antagonist DNA. However, the recovery ofgenomic DNA encoding the antagonist is more complex than that of anexogenously replicated vector because restriction enzyme digestion isrequired to excise the antagonist DNA.

(iii) Selection Gene Component

Expression and cloning vectors typically contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin [Southern et al., J. Molec. Appl. Genet., 1:327(1982)], mycophenolic acid (Mulligan et al., Science, 209:1422 (1980)]or hygromycin [Sugden et al., Mol. Cell. Biol., 5:410-413 (1985)]. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantagonist nucleic acid, such as DHFR or thymidine kinase. The mammaliancell transformants are placed under selection pressure that only thetransformants are uniquely adapted to survive by virtue of having takenup the marker. Selection pressure is imposed by culturing thetransformants under conditions in which the concentration of selectionagent in the medium is successively changed, thereby leading toamplification of both the selection gene and the DNA that encodes thereceptor antagonist. Amplification is the process by which genes ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Other examples of amplifiable genes includemetallothionein-I and -II, adenosine deaminase, and ornithinedecarboxylase.

Cells transformed with the DHFR selection gene may first be identifiedby culturing all of the transformants in a culture medium that containsmethotrexate (Mtx), a competitive antagonist of DHFR. An appropriatehost cell when wild-type DHFR is employed is the Chinese hamster ovary(CHO) cell line deficient in DHFR activity, prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).The transformed cells are then exposed to increased levels ofmethotrexate. This leads to the synthesis of multiple copies of the DHFRgene, and, concomitantly, multiple copies of other DNA comprising theexpression vectors, such as the DNA encoding the receptor antagonist.This amplification technique can be used with any otherwise suitablehost, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding HGF receptor antagonist, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)]. The presence of thetrp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts [Bianchi et al.,Curr. Genet., 12:185 (1987)]. More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis [Van den Berg, Bio/Technology, 8:135 (1990)]. Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed[Fleer et al., Bio/Technology, 9:968-975 (1991)].

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the receptorantagonist nucleic acid sequence. Promoters are untranslated sequenceslocated upstream (5′) to the start codon of a structural gene (generallywithin about 100 to 1000 bp) that control the transcription andtranslation of particular nucleic acid sequence to which they areoperably linked. Such promoters typically fall into two classes,inducible and constitutive. Inducible promoters are promoters thatinitiate increased levels of transcription from DNA under their controlin response to some change in culture conditions, e.g., the presence orabsence of a nutrient or a change in temperature. At this time a largenumber of promoters recognized by a variety of potential host cells arewell known. These promoters are operably linked to receptor antagonistencoding DNA by removing the promoter from the source DNA by restrictionenzyme digestion and inserting the isolated promoter sequence into thevector. Various heterologous promoters may be used to directamplification and/or expression of the receptor antagonist DNA.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems [Chang et al., Nature, 275:615(1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, atryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057(1980); EP 36,776], and hybrid promoters such as the tac promoter[deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. However,other known bacterial promoters are suitable. Their nucleotide sequenceshave been published, thereby enabling a skilled worker operably toligate them to DNA encoding receptor antagonist [Siebenlist et al.,Cell, 20:269 (1980)] using linkers or adaptors to supply any requiredrestriction sites. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding HGF receptor antagonist.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. At the 3′ endof most eukaryotic genes is an AATAAA sequence that may be the signalfor addition of the poly A tail to the 3′ end of the coding sequence.All of these sequences are suitably inserted into eukaryotic expressionvectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

HGF receptor antagonist transcription from vectors in mammalian hostcells is controlled, for example, by promoters obtained from the genomesof viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand most preferably Simian Virus 40 (SV40), or from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication [Fiers et al., Nature, 273:113 (1978); Mulligan and Berg,Science, 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci.USA, 78:7398-7402 (1981)]. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment [Greenaway et al., Gene, 18:355-360 (1982)]. A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978 [See also Gray et al.,Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferonin monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus; Canaani and Berg,Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of thehuman interferon 131 gene in cultured mouse and rabbit cells; and Gormanet al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression ofbacterial CAT sequences in CV-1 monkey kidney cells, chicken embryofibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3cells using the Rous sarcoma virus long terminal repeat as a promoter].

(v) Enhancer Element Component

Transcription of a DNA encoding the HGF receptor antagonist of thisinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Enhancers are relatively orientation and positionindependent, having been found 5′ [Laimins et al., Proc. Natl. Acad.Sci. USA, 78:993 (1981]) and 3′ [Lusky et al., Mol. Cell. Bio., 3:1108(1983]) to the transcription unit, within an intron [Banerji et al.,Cell, 33:729 (1983)], as well as within the coding sequence itself[Osborne et al., Mol. Cell. Bio., 4:1293 (1984)]. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature, 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the HGF receptor antagonist-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding HGF receptor antagonist.

(vii) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures can be used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9:309 (1981) or by the method of Maxam et al., Methods inEnzymology, 65:499 (1980).

(viii) Transient Expression Vectors

Expression vectors that provide for the transient expression inmammalian cells of DNA encoding HGF receptor antagonist may be employed.In general, transient expression involves the use of an expressionvector that is able to replicate efficiently in a host cell, such thatthe host cell accumulates many copies of the expression vector and, inturn, synthesizes high levels of a desired polypeptide encoded by theexpression vector [Sambrook et al., supra]. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of the receptor antagonists.

(ix) Suitable Exemplary Vertebrate Cell Vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of HGF receptor antagonist in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

3. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include but are not limitedto eubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis (e.g., B.licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonas such as P. aeruginosa, and Streptomyces. Preferably, thehost cell should secrete minimal amounts of proteolytic enzymes.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for HGF receptorantagonist-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein.

Suitable host cells for the expression of glycosylated HGF receptorantagonist are derived from multicellular organisms.

Such host cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified [See, e.g., Luckow etal., Bio/Technology, 6:47-55 (1988); Miller et al., in GeneticEngineering, Setlow et al., eds., Vol. 8 (Plenum Publishing, 1986), pp.277-279; and Maeda et al., Nature, 315:592-594 (1985)]. A variety ofviral strains for transfection are publicly available, e.g., the L-1variant of Autographa californica NPV and the Bm-5 strain of Bombyx moriNPV.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the receptor antagonist-encoding DNA. During incubation of theplant cell culture with A. tumefaciens, the DNA encoding the receptorantagonist is transferred to the plant cell host such that it istransfected, and will, under appropriate conditions, express thereceptor antagonist-encoding DNA. In addition, regulatory and signalsequences compatible with plant cells are available, such as thenopaline synthase promoter and polyadenylation signal sequences[Depicker et al., J. Mol. Appl. Gen., 1:561 (1982)]. In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue [EP321,196 published 21 Jun. 1989].

Propagation of vertebrate cells in culture (tissue culture) is also wellknown in the art [See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973)]. Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; and FS4 cells.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for HGF receptorantagonist production and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in Sambrook et al., supra, orelectroporation is generally used for prokaryotes or other cells thatcontain substantial cell-wall barriers. Infection with Agrobacteriumtumefaciens is used for transformation of certain plant cells, asdescribed by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published29 Jun. 1989. In addition, plants may be transfected using ultrasoundtreatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) is preferred. General aspects of mammalian cell host systemtransformations have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

4. Culturing the Host Cells

Prokaryotic cells used to produce HGF receptor antagonist may becultured in suitable media as described generally in Sambrook et al.,supra.

The mammalian host cells used to produce HGF receptor antagonist may becultured in a variety of media. Examples of commercially available mediainclude Ham's F10 (Sigma), Minimal Essential Medium (“MEM”, Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (“DMEM”,Sigma). Any such media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, and particularly ³²P. However, other techniques may alsobe employed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionucleotides, fluorescers or enzymes. Alternatively,antibodies may be employed that can recognize specific duplexes,including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The antibodies in turn may be labeled and theassay may be carried out where the duplex is bound to a surface, so thatupon the formation of duplex on the surface, the presence of antibodybound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. With immunohistochemicalstaining techniques, a cell sample is prepared, typically by dehydrationand fixation, followed by reaction with labeled antibodies specific forthe gene product coupled, where the labels are usually visuallydetectable, such as enzymatic labels, fluorescent labels, or luminescentlabels. Antibodies useful for immunohistochemical staining and/or assayof sample fluids may be either monoclonal or polyclonal, and may beprepared in any mammal.

6. Purification of HGF Receptor Antagonist Polypeptide

HGF receptor antagonist preferably is recovered from the culture mediumas a secreted polypeptide, although it also may be recovered from hostcell lysates when directly produced without a secretory signal. If thereceptor antagonist is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or itsextracellular region may be released by enzymatic cleavage.

When the antagonist is produced in a recombinant cell other than one ofhuman origin, the antagonist polypeptide is free of proteins orpolypeptides of human origin. However, it may be desired to purify thereceptor antagonist from recombinant cell proteins or polypeptides toobtain preparations that are substantially homogeneous as to thereceptor antagonist. As a first step, the culture medium or lysate maybe centrifuged to remove particulate cell debris. HGF receptorantagonist thereafter is purified from contaminant soluble proteins andpolypeptides, with the following procedures being exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; and protein A Sepharose columns to remove contaminantssuch as IgG.

A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants.

7. Covalent Modifications

Covalent modifications of HGF receptor antagonists are included withinthe scope of this invention. One type of covalent modification of theHGF receptor antagonist is introduced into the molecule by reactingtargeted amino acid residues of the antagonist with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the antagonist.

Derivatization with bifunctional agents is useful for crosslinking theantagonist to a water-insoluble support matrix or surface for use in amethod for purifying. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [I.E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group. The modifiedforms of the residues fall within the scope of the present invention.

Another type of covalent modification of the receptor antagonistpolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties and/or adding one ormore glycosylation sites that are not present in the native polypeptide.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-Z-serine and asparagine-Z-threonine, where Z is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the polypeptide may be accomplishedby altering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to the nativesequence (for O-linked glycosylation sites). The amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the polypeptide at preselected bases such thatcodons are generated that will translate into the desired amino acids.The DNA mutation(s) may be made using methods described above and inU.S. Pat. No. 5,364,934, supra.

Another means of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published September 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. For instance, chemical deglycosylation by exposing thepolypeptide to the compound trifluoromethanesulfonic acid, or anequivalent compound can result in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the polypeptide intact. Chemical deglycosylation isdescribed by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987)and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavageof carbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol., 138:350 (1987).

Glycosylation at potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duskin et al., J. Biol.Chem., 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification comprises linking the HGF receptorantagonist to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes,in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. WO 93/00109 also describesmethods of linking amino acid residues in polypeptides to PEG molecules.

8. HGF Receptor Antagonist Chimeras

The present invention also provides chimeric molecules comprising HGFreceptor antagonist fused to another, heterologous polypeptide.

In one embodiment, the chimeric molecule comprises a fusion of theantagonist with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the antagonist. Thepresence of such epitope-tagged forms of the antagonist can be detectedusing an antibody against the tag polypeptide. Also, provision of theepitope tag enables the antagonist to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include the flu HA tag polypeptide and its antibody12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myctag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan etal., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptidesinclude the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210(1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194(1992)]; an α-tubulin epitope peptide [Skinner et al., J. Biol. Chem.,266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)]. Once the tag polypeptide has been selected, an antibody theretocan be generated using the techniques disclosed herein.

Generally, epitope-tagged antagonist may be constructed and producedaccording to the methods described above. HGF receptor antagonist-tagpolypeptide fusions are preferably constructed by fusing the cDNAsequence encoding the HGF receptor antagonist portion in-frame to thetag polypeptide DNA sequence and expressing the resultant DNA fusionconstruct in appropriate host cells. Ordinarily, when preparing the HGFreceptor antagonist-tag polypeptide chimeras of the present invention,nucleic acid encoding the antagonist will be fused at its 3′ end tonucleic acid encoding the N-terminus of the tag polypeptide, however 5′fusions are also possible. For example, a polyhistidine sequence ofabout 5 to about 10 histidine residues may be fused at the N-terminus orthe C-terminus and used as a purification handle in affinitychromatography.

Epitope-tagged HGF receptor antagonist can be purified by affinitychromatography using the anti-tag antibody. The matrix to which theaffinity antibody is attached may include, for instance, agarose,controlled pore glass or poly(styrenedivinyl)benzene. The epitope-taggedHGF receptor antagonist can then be eluted from the affinity columnusing techniques known in the art.

In another embodiment of the invention, the HGF receptor antagonist maybe fused to a salvage receptor binding epitope in order to increase itsserum half-life. This may be achieved, for example, by incorporation ofa salvage receptor binding epitope into a HGF receptor antagonistantibody fragment (e.g., by mutation of the appropriate region in theantibody fragment or by incorporating the epitope into a peptide tagthat is then fused to the antibody fragment at either the end or in themiddle, e.g., by DNA or peptide synthesis).

A systematic method for preparing such a chimera having an increased invivo half-life comprises several steps. The first involves identifyingthe sequence and conformation of a salvage receptor binding epitope ofan Fc region of an IgG molecule. Once this epitope is identified, thesequence of the HGF receptor antagonist of interest is modified toinclude the sequence and conformation of the identified binding epitope.After the sequence is mutated, the chimera is tested to see if it has alonger in vivo half-life than that of the original antagonist. If thechimera does not have a longer half-life upon testing, its sequence isfurther altered to include the sequence and conformation of theidentified binding epitope.

The salvage receptor binding epitope being incorporated into the HGFreceptor antagonist of interest is any suitable such epitope as definedabove, and its nature will depend for example, on the type of antagonistbeing modified. The transfer is made such that the HGF receptorantagonist of interest still possesses antagonistic activity.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain and is preferably transferred to an analogous position in anHGF receptor antagonist antibody fragment. Preferably, three or moreresidues from one or two loops of the Fc domain are transferred, andmore preferably, the epitope is taken from the CH2 domain of the Fcregion of an IgG and transferred to the CH1, CH3, or V_(H) region, ormore than one such region, of the antagonist antibody. Alternatively,the epitope is taken from the CH2 domain of the Fc region andtransferred to the C_(L) region or the V_(L) region, or both, of theantagonist antibody fragment.

In another embodiment, the chimeric molecule comprises a HGF receptorantagonist fused to an immunoglobulin constant domain or anotherheterologous polypeptide such as albumin. This includes chimeras inmonomeric, homo- or heteromultimeric form, and particularlyheterodimeric form.

In general, the chimeric molecules can be constructed in a fashionsimilar to chimeric antibodies in which a variable domain from anantibody of one species is substituted for the variable domain ofanother species. See, for example, EP 0 125 023; EP 173,494; Munro,Nature, 312:597 (13 Dec. 1984); Neuberger et al., Nature, 312:604-608(13 Dec. 1984); Sharon et al., Nature, 309:364-367 (24 May 1984);Morrison et al., Proc. Nat'l. Acad. Sci. USA, 81:6851-6855 (1984);Morrison et al., Science, 229:1202-1207 (1985); Boulianne et al.,Nature, 312:643-646 (13 Dec. 1984); Capon et al., Nature, 337:525-531(1989); Traunecker et al., Nature, 339:68-70 (1989). Preferably, the Igis a human immunoglobulin when the chimera is intended for in vivotherapy for humans. DNA encoding immunoglobulin light or heavy chainconstant regions is known or readily available from cDNA libraries orcan be synthesized. See for example, Adams et al., Biochemistry,19:2711-2719 (1980); Gough et al., Biochemistry, 19:2702-2710 (1980);Dolby et al., Proc. Natl. Acad. Sci. USA, 77:6027-6031 (1980); Rice etal., Proc. Natl. Acad. Sci., 79:7862-7865 (1982); Falkner et al.,Nature, 298:286-288 (1982); and Morrison et al., Ann. Rev. Immunol.,2:239-256 (1984).

Further details of how to prepare such fusions are found in publicationsconcerning the preparation of immunoadhesins. Immunoadhesins in general,and CD4-Ig fusion molecules specifically, are disclosed in WO 89/02922,published 6 Apr. 1989. Molecules comprising the extracellular portion ofCD4, the receptor for human immunodeficiency virus (HIV), linked to IgGheavy chain constant region are known in the art and have been found tohave a markedly longer half-life and lower clearance than the solubleextracellular portion of CD4 [Capon et al., supra; Byrn et al., Nature,344:667 (1990)].

In another embodiment, the chimera comprises a HGF receptor antagonistfused to albumin. Such chimeras may be constructed by inserting theentire coding region of albumin into a plasmid expression vector. TheDNA encoding the antagonist can be inserted at the 5′ end of the albuminsequence, along with an insert encoding a linker consisting of fourglycine residues [Lu et al., FEBS Letters, 356:56-59 (1994)]. The HGFreceptor antagonist-albumin chimera can then be expressed in desiredmammalian cells or yeast, for instance.

C. Methods of Treatment and Diagnosis

In another embodiment of the invention, methods for treating cancer areprovided. In the methods, HGF receptor antagonist is administered to amammal diagnosed as having cancer. While the term “cancer” as usedherein is not limited to any one specific form of the disease, it isbelieved that the methods will be particularly effective for cancerswhich are found to be accompanied by increased levels of HGF, oroverexpression or activation of HGF receptor in the mammal. In apreferred method of the invention, the cancer is breast cancer. It is ofcourse contemplated that the methods of the invention can be employed incombination with still other therapeutic techniques such as surgery.

The antagonist is preferably administered to the mammal in apharmaceutically-acceptable carrier. Suitable carriers and theirformulations are described in Remington's Pharmaceutical Sciences, 16thed., 1980, Mack Publishing Co., edited by Oslo et al. Typically, anappropriate amount of a pharmaceutically-acceptable salt is used in theformulation to render the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antagonist, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antagonistbeing administered.

The antagonist can be administered to the mammal by injection (e.g.,intravenous, intraperitoneal, subcutaneous, intramuscular), or by othermethods such as infusion that ensure its delivery to the bloodstream inan effective form. The antagonist may also be administered byintratumoral, peritumoral, intralesional, or perilesional routes, toexert local as well as systemic therapeutic effects. Local orintravenous injection is preferred.

Effective dosages and schedules for administering the antagonist may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antagonist that must be administered will vary depending on,for example, the mammal which will receive the antagonist, the route ofadministration, the particular type of antagonist used and other drugsbeing administered to the mammal. Guidance in selecting appropriatedoses for antibody antagonists is found in the literature on therapeuticuses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone etal., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp.303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haberet al., eds., Raven Press, New York (1977) pp. 365-389. A typical dailydosage of the antagonist used alone might range from about 1 μg/kg to upto 100 mg/kg of body weight or more per day, depending on the factorsmentioned above.

The antagonist may also be administered to the mammal in combinationwith effective amounts of one or more other therapeutic agents or inconjunction with radiation treatment. Therapeutic agents contemplatedinclude chemotherapeutics as well as immunoadjuvants and cytokines.Chemotherapies contemplated by the invention include chemical substancesor drugs which are known in the art and are commercially available, suchas Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate,Cisplatin, Melphalan, Vinblastine and Carboplatin. The antagonist may beadministered sequentially or concurrently with the one or more othertherapeutic agents. The amounts of antagonist and therapeutic agentdepend, for example, on what type of drugs are used, the cancer beingtreated, and the scheduling and routes of administration but wouldgenerally be less than if each were used individually.

Following administration of antagonist to the mammal, the mammal'scancer and physiological condition can be monitored in various ways wellknown to the skilled practitioner. For instance, tumor mass may beobserved physically or by standard x-ray imaging techniques.

The antagonists of the invention also have utility in non-therapeuticapplications. For instance, methods for employing the antagonists invitro in diagnostic assays are provided. For instance, the antagonistsmay be employed in diagnostic assays to detect overexpression of HGFreceptor in specific cells and tissues. Various diagnostic assaytechniques known in the art may be used, such as competitive bindingassays, direct or indirect sandwich assays and immunoprecipitationassays conducted in either heterogeneous or homogeneous phases [Zola,Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987)pp. 147-158]. The antagonists used in the diagnostic assays can belabeled with a detectable moiety. The detectable moiety should becapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin,or an enzyme, such as alkaline phosphatase, beta-galactosidase orhorseradish peroxidase. Any method known in the art for conjugating theantagonist to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature, 194:495 (1962); David etal., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth.,40:219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30:407(1982).

Additionally, the HGF receptor antagonist antibodies can be used toimmunopurify HGF receptor(s).

D. Articles of Manufacture and Kits

In a further embodiment of the invention, there are provided articles ofmanufacture and kits containing materials useful for treating cancer ordetecting or purifying HGF receptor. The article of manufacturecomprises a container with a label. Suitable containers include, forexample, bottles, vials, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition having an active agent which is effective fortreating cancer or for detecting or purifying HGF receptor. The activeagent in the composition is a HGF receptor antagonist and preferably,comprises Fab fragments of monoclonal antibodies specific for c-Met. Thelabel on the container indicates that the composition is used fortreating cancer or detecting or purifying HGF receptor, and may alsoindicate directions for either in vivo or in vitro use, such as thosedescribed above.

The kit of the invention comprises the container described above and asecond container comprising a buffer. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All reference citations herein areincorporated by reference.

EXAMPLES

All restriction enzymes referred to in the examples were purchased fromNew England Biolabs and used according to manufacturer's instructions.All other commercially available reagents referred to in the exampleswere used according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Rockville, Md.

Example 1 Preparation of anti-c-Met Antibodies

Balb/c mice (obtained from Charles River Laboratories) were immunized byinjecting 2.5 μg/50 μl c-Met-IgG fusion protein (diluted in MPL-TDMadjuvant purchased from Ribi Immunochemical Research Inc., Hamilton,Mont.) five times into each hind foot pad. Injections were administeredon Day 0 and Days 56, 63, 66 and 73. The c-Met-IgG fusion protein(including the extracellular domain of c-Met fused to a human IgG1 heavychain) was constructed essentially as described by Mark et al., J. Biol.Chem., 267:26166-26171 (1992) and produced in Chinese hamster ovary(CHO) cells. The c-Met-IgG was subsequently purified in a single stepusing affinity chromatography on immobilized Protein A (Bioprocessing,Inc., Princeton, N.J.), using an elution scheme modified from Chamow etal., J. Immunol., 153:4268-4280 (1994). Culture supernatant was loadedonto a Protein A column equilibrated in 20 mM Tris, pH 7.4, 0.15 M NaCl.The column was washed, first with equilibration buffer, then withequilibration buffer containing 0.5 M tetramethylammonium chloride, toremove non-specifically bound protein. c-Met-IgG was eluted with 20 mMTris, pH 7.4, 3.5 M MgCl₂. This c-Met-IgG eluate was concentrated andexchanged into 20 mM Tris, pH 7.4, 0.15 M NaCl by gel filtration onSephadex G25 to a final concentration of about 2-4 mg/ml.

On Day 77, popliteal lymph nodes were removed from the mice and a singlecell suspension was prepared in DMEM media (obtained from BiowhitakkerCorp.) supplemented with 1% penicillin-streptomycin. The lymph nodecells were then fused with murine myeloma cells P3X63AgU.1 (ATCC CRL1597) using 35% polyethylene glycol and cultured in 96-well cultureplates. Hybridomas resulting from the fusion were selected in HATmedium. Ten days after the fusion, hybridoma culture supernatants werescreened in an ELISA to test for the presence of monoclonal antibodiesbinding to the c-Met-IgG fusion protein.

In the ELISA, 96-well microtiter plates (Nunc) were coated by adding 50μl of 2 μg/ml goat anti-human IgG Fc (purchased from CappelLaboratories) to each well and incubating at 4° C. overnight. The plateswere then washed three times with distilled water. The wells in themicrotiter plates were blocked with 200 μl of 2% bovine serum albuminand incubated at room temperature for 1 hour. The plates were thenwashed again three times with distilled water.

After the washing step, 100 μl of 0.4 μg/ml c-Met-IgG fusion protein (asdescribed above) was added to each well. The plates were incubated for 1hour at room temperature on a shaker apparatus, followed by washingthree times with distilled water.

Then, 100 μl of the hybridoma supernatants was added to designatedwells. 100 μl of P3X63AgU.1 myeloma cell conditioned medium was added toother designated wells as controls. The plates were incubated at roomtemperature for 1 hour on a shaker apparatus and then washed three timeswith distilled water.

Next, 50 μl HRP-conjugated goat anti-mouse IgG Fc (purchased from CappelLaboratories), diluted 1:1000 in assay buffer (0.5% bovine serumalbumin, 0.05% Tween-20, 0.01% Thimersol in PBS), was added to each welland the plates incubated for 1 hour at room temperature on a shakerapparatus. The plates were washed three times with distilled water,followed by addition of 50 μl of substrate (5 mg OPD, 12.5 ml PBS, 5 μlH₂O₂) to each well and incubation at room temperature for 10 minutes.The reaction was stopped by adding 50 μl of 2 N H₂SO₄ to each well, andabsorbance at 490 nm was read in an automated microtiter plate reader.

Of 912 hybridoma supernatants screened in the ELISA, 24 supernatantstested positive (calculated as approximately 2 times above background).The supernatants testing positive in the ELISA were further analyzed byFACS analysis using A549 cells (human epidermoid cell line expressingc-Met; ATCC CCL 185) or BaF3 transfected cells expressing c-Met (seeExample 6 below) and fluorescein-conjugated mouse anti-IgG. The FACSanalysis showed 19/24 supernatants were positive for anti-c-Metantibodies.

Example 2 Preparation of anti-c-Met Antibody 5D5.11.6

Balb/c mice were immunized as described in Example 1 except that thec-Met-IgG fusion protein injections were administered on Day 0 and Days7, 14, 21, 28, 211, 273 and 279. On Day 282, lymph nodes were removedand a fusion was conducted as described in Example 1. Hybridomasupernatants were tested according to the ELISA described in Example 1.One of the positive anti-c-Met monoclonal antibodies was called 5D5.11.6(“5D5”). Further antibody analysis showed that the 5D5 monoclonalantibody is an IgG1 isotype antibody comprising a kappa light chain.

Ascites was produced in Balb/c mice and the monoclonal antibodies werethen purified using a protein G affinity column. The proteinconcentration was determined by the absorbance at 280 nm using anextinction coefficient of 1.4.

Example 3 Inhibition Assay of anti-c-Met Antibody 1A3.3.13 to Block HGFBinding

An inhibition assay was conducted to examine the ability of theantibodies (described in Example 1) to block binding of HGF to c-Met-IgGfusion protein. Prior to conducting the inhibition assay, the 24hybridoma supernatants determined positive in the ELISA in Example 1were purified on Protein A-Sepharose columns to yield antibodypreparations of about 1 μg/ml.

For the assay, 96-well microtiter plates were coated by adding 100 μl of2 μg/ml goat anti-human Fc (purchased from Jackson Immunochemical, WestGrove, Pa.) in 0.05 M sodium bicarbonate, pH 9.6, to each well andincubating overnight at 4° C. The plates were then washed with a washingbuffer (0.05% Tween-20, 0.01% Thimersol in PBS). Nonspecific binding inthe wells was blocked by adding 150 μl blocking buffer (0.5% BSA, 0.01%Thimersol in PBS, pH 7.4) to each well and incubating at roomtemperature for 2 hours with rapid agitation on an orbital shaker.

Next, continuing at room temperature and agitation on the orbitalshaker, the blocking buffer was removed from the wells, and the plateswere washed with washing buffer. Next, 100 μl of 10 ng/ml c-Met-IgGfusion protein in PBS, 0.5% BSA, 0.05% Tween-20, and 0.01% Thimersol(described in Example 1) was added to the wells. The plates wereincubated for 2 hours and then washed 3 times with washing buffer.

Recombinant human HGF (rhuHGF) was produced in CHO cells using aprocedure modified from Naka et al., J. Biol. Chem., 267:20114-20119(1992). rhuHGF-transfected cells were grown in a 400 L bioreactor inmedium containing 2% fetal bovine serum for 8 days. Culture supernatantcontaining rhuHGF was concentrated and clarified, then conditioned bythe addition of solid NaCl to 0.3 M. rhuHGF was then purified in asingle step using cation exchange chromatography. Conditioned,concentrated culture supernatant was loaded onto a column of S-SepharoseFast Flow equilibrated in 20 mM Tris, pH 7.5, 0.3 M NaCl. After washingout unbound protein, rhuHGF was eluted in a linear gradient from 20 mMTris, pH 7.5, 0.3 M NaCl to 20 mM Tris, pH 7.5, 1.2 M NaCl.rhuHGF-containing fractions were pooled based on SDS-PAGE analysis. TheS Sepharose Fast Flow pool was concentrated and exchanged into 20 mMTris, pH 7.5, 0.5 M NaCl by gel filtration on Sephadex G25 to a finalconcentration of about 3-5 mg/ml. A rhuHGF stock solution was thenprepared by diluting the rhuHGF in assay buffer (0.5% bovine serumalbumin, 0.05% Tween-20, 0.01% Thimersol in PBS) to a concentration of10 μg/ml. A stock solution of control gp120 antibody (Genentech, Inc.)was also prepared by diluting the antibody in assay buffer to aconcentration of 10 μg/ml.

Then, 50 μl of either rhuHGF, gp120 antibody or one of the 24 monoclonalantibodies was added to designated wells to yield a final concentrationof 1000, 100, 10, or 1 ng/ml/well. Immediately, 50 μl of 200 ng/mlbiotinylated rhuHGF (rhuHGF biotinylated using Biotin-X-NHS obtainedfrom Research Organics, Inc., Cleveland, Ohio) was also added to eachwell. After 2 hours incubation, the wells were washed 3 times withwashing buffer. 100 μl HRP-streptavidin (1:2000 dilution in assaybuffer) (purchased from Zymed Laboratories), was added to the wells andthe plates were incubated for 30 minutes. The plates were washed again 3times with washing buffer. 50 μl of substrate (5 mg OPD, 12.5 ml PBS, 5μl H₂O₂) was added to the wells and color was allowed to develop for 20minutes. The reaction was stopped by adding 100 μl 4.5 N sulfuric acidto each well. Absorbance at 492 nm was quantitated in an automatedmicrotiter plate reader.

One of the monoclonal antibodies, referred to as 1A3.3.13, significantlyblocked binding of HGF (FIG. 2). Further antibody analysis showed thatthe 1A3.3.13 monoclonal antibody is an IgG1 isotype antibody comprisinga kappa light chain.

Example 4 Mitogenic Assay of Antibodies 3D6, 6G1 and 1A3.3.13 on HumanMammary Epithelial Cell Line

Several different antibodies produced in the fusion described in Example1, referred to as 3D6, 6G1, and 1A3.3.13, and purified as described inExample 3, were tested and compared for their ability to induce DNAsynthesis in a human mammary epithelial cell bioassay.

Human mammary epithelial cells (obtained from Clonetics Corp., No.CC-2551) were passaged in Mammary Epithelial Cell Basal Medium(Clonetics Corp., No. CC-3151). Prior to conducting the bioassay, thecells were trypsinized, washed, and resuspended in assay medium (BasalMedium supplemented with 1 mg/ml BSA, penicillin, streptomycin andL-glutamine) to a concentration of 1×10⁵ cells/ml. Next, 100 μl of thecells were added to the wells of 96-well culture plates. rhuHGF(described in Example 3) was diluted in assay medium at concentrationsof 20 ng/ml and 200 ng/ml. The 3D6, 6G1, 1A3.3.13, and control gp120antibodies were diluted in assay medium at concentrations of 200 ng/mland 2 μg/ml. 100 μl of the rhuHGF and antibody preparations was thenadded to designated wells. The plates were incubated at 37° C. in 5% CO₂for 16 hours.

Next, 1 μCi ³H-thymidine (Amersham) was added to each well, and theplates were incubated for 24 hours at 37° and 5% CO₂. The human mammaryepithelial cells were harvested and the amount of radioactivityincorporated into the DNA was then quantitated in a microplatescintillation counter.

The results showed that, at a concentration of 10 ng/ml, the 3D6, 6G1and 1A3.3.12 antibodies have some HGF agonistic effect. (See FIG. 3)

Example 5 Mitogenic Assay of Antibodies 05-237 and 05-238 on Mink LungCell Line

Anti-c-Met antibodies 05-237 and 05-238 (purchased from UpstateBiotechnology Inc., Lake Placid, N.Y.; see also, Prat et al., Mol. Cell.Biol., supra, Prat et al., Int. J. Cancer, supra) and antibody 3D6(described in Example 4) were tested and compared for their ability toinduce DNA synthesis in a mink lung bioassay.

Mink lung cells (Mv 1 Lu, ATCC CCl 64) were passaged in DME/F12 (50:50)supplemented with 10% fetal bovine serum, penicillin, streptomycin andL-glutamine. Prior to conducting the bioassay, the mink lung cells weretrypsinized, washed, and resuspended in assay medium (DME/F12 mediumsupplemented with 1 mg/ml BSA, penicillin, streptomycin and L-glutamine)to a concentration of 1×10⁵ cells/ml. The bioassay was then conducted asdescribed in Example 4.

The results showed that antibodies 05-237 and 05-238 have HGF agonisticeffect. (See FIG. 4)

Example 6 Antagonistic Activity of Monoclonal Antibody 1A3.3.13 Fab

Antagonistic activity of 1A3.3.13 monoclonal antibody Fab fragments wasdetermined using a thymidine incorporation assay. Monoclonal antibody1A3.3.13 (described in Example 3) was digested with papain to obtain theFab fragments. The papain digestion was performed by initially dialyzingthe antibody against a 20 mM phosphate/10 mM EDTA, pH 7.0, bufferovernight. The antibody was then concentrated to approximately 10 mg/ml.Next, 0.5 ml immobilized papain (crosslinked 6% beaded agarose, obtainedfrom Pierce Chemicals) was added to a 16×100 mm tube. The papain beadswere washed 2 times with 4 ml of digestion buffer (42 mg cysteine-HCl in12 ml phosphate buffer, pH 10). Each wash was removed using a separator.About 0.5 to 1 ml of the 1A3.3.13 antibody was added to the papain beadsand then incubated in a heated shaker bath (37° C., 200 rpm) overnight.1.5 ml of binding buffer (Immunopure IgG Binding Buffer obtained fromPierce Chemicals) was added to the tube, and the supernatant wasseparated from the beads with a separator. The supernatant was thenpassed over a Protein A column equilibrated with the binding buffer.Additional binding buffer was passed over the column and the eluatecontaining the Fab fragments was collected in 1 ml fractions. Thefractions were analyzed by absorbance at 280 nm and the Fab containingfractions dialyzed against PBS overnight. Absorbance at 280 nm was readagain to determine the concentration of Fab (about 1.53). The fractionswere also run on a 7.5% SDS gel to determine the purity of the Fab inthe fraction. The 1A3.3.13 Fab fragments were further tested in aninhibition assay (as described in Example 3). The Fab fragments didinhibit HGF binding but showed a weaker inhibitory effect as compared tointact 1A3.3.13 antibody (data not shown).

An expression plasmid was prepared by inserting a full-length cDNA forhuman c-Met (described as pOK met cDNA in Rodrigues et al., supra) intoa pRK5.tk.neo vector [de Sauvage et al., Nature, 369:533-538 (1994);Gorman, DNA Cloning: A New Approach, 2:143-190 (IRL Washington 1985)].The resulting plasmid was linearized and transfected into the IL-3dependent cell line, BaF3 [Palacios et al., Cell, 41:727-734 (1985)] byelectroporation (800 microfarad, 250 V, BRL electroporator). Selectionof transfectants was performed by culturing the cells for 2-3 weeks inthe presence of 2 mg/ml G418. One of the selected transfectant celllines, referred to as BaF3-hmet.8, was confirmed by Western blotting toexpress c-Met. BaF3-hmet.8 also tested positive for response to HGF in aproliferation assay measuring incorporation of ³H-thymidine. Neither theparental BaF3 cells nor any cells derived by transfection with thepRK5.tk.neo vector alone (“BaF3-neo”) were found to express c-Met orrespond to HGF in the proliferation assay.

The BaF3-hmet.8 cells were passaged in RPMI medium supplemented with 10%fetal bovine serum, 5% WEHI-conditioned medium (as a source of IL-3) and2 mM glutamine. Prior to conducting the assay, the cells were washedtwice with assay medium (RPMI medium supplemented with 10% fetal bovineserum) and resuspended in assay medium to a concentration of 5×10⁴cells/ml. Next, 100 μl of the cells was added to each well in the96-well culture plates. Various concentrations (0.2 μg/ml, 2 μg/ml, 20μg/ml) of control gp120 Fab fragments (gp120 monoclonal antibodydigested with papain as described above) and the 1A3.3.13 Fab fragmentswere prepared in assay medium and 100 μl was added to designated wells.The plates were incubated at 37° C. in 5% CO₂ for 15 hours.

One μCi ³H-thymidine was added to each well of the culture plates. Thecells were harvested 7 hours later and the amount of radioactivityincorporated into the DNA was quantitated (CPM) in a microplatescintillation counter.

The results are shown in FIG. 5. At a concentration of 10 μg/ml, the1A3.3.13 Fab fragments significantly blocked BaF3-hmet.8 cellproliferation in the presence of HGF.

Example 7 Preparation of 5D5 Antibody Fab

The 5D5 monoclonal antibody (as described in Example 2) was dialyzed anddigested with papain essentially as described in Example 6 except thatafter dialysis, the antibody was concentrated to 7 mg/ml using aCentricon 30 filter. After dialyzing the 5D5 Fab fragments against PBSovernight, the preparation of 5D5 Fab fragments was further purified bygel filtration (Superose™ 12, Pharmacia) to remove residual F(ab′)₂.

Example 8 Assay of 5D5 Antibody and 5D5 Fab Binding to c-Met

The binding specificity of the 5D5 antibody (described in Example 2) wasexamined by incubating BaF3-hmet cells or BaF3-neo cells (each describedin Example 6) with saturating concentrations of 5D5 antibody or acontrol IgG, followed by fluorescein-conjugated mouse anti-IgG. 10 μg/ml5D5 antibody was incubated with 50 μl of 1×10⁵ BaF3-hmet cells orBaF3-neo cells for 30 minutes at 4° C. in cell sorting buffer (PBS and1% fetal calf serum). The cells were washed twice with cell sortingbuffer and spun at 1500 rpm for 5 minutes. The cells were then incubatedwith 100 μl goat (Fab′)₂ anti-mouse IgG Fc (Cappel) at a 1:1000 dilutionfor 30 minutes at 4° C. The cells were again washed twice with cellsorting buffer and spun at 1500 rpm for 5 minutes. The cells were thentransferred to microtiter tubes with 250 μl of cell sorting buffer andanalyzed by flow cytometry with a Becton Dickinson FACScan. As shown inFIGS. 6A and 6B, the 5D5 antibody binds to the BaF3-hmet cells but notto the BaF3-neo cells, indicating that 5D5 antibody binds c-Met.

An inhibition assay was also conducted, essentially as described inExample 3, to examine the ability of 5D5 antibody (Example 2) and 5D5Fab (described in Example 7) to block binding of HGF to c-Met-IgG fusionprotein. rhuHGF, 5D5 antibody, 5D5 Fab and control gp120 Fab were testedat concentrations ranging from 0 to 10 μg/ml, as shown in FIG. 7. Eachdata point in the graph of FIG. 7 is the mean of triplicates. The dataillustrated in FIG. 7 shows that both the 5D5 antibody and 5D5 Fabblocked binding of HGF to c-Met-IgG.

Example 9 Antagonistic Activity of 5D5 Fab

A. BaF3-hmet Cell Assay

Antagonistic activity of 5D5 Fab fragments was examined using athymidine incorporation assay, as described in Example 6. Variousconcentrations (0, 0.01, 0.1, 1, 10 μg/ml) of control gp 120 Fab and 5D5Fab (Example 7) were prepared in the assay medium and added todesignated wells, either alone or in the presence of 10 ng/ml rhuHGF.The results are shown in FIGS. 8A and 8B. Data are the mean±SEM of 4replicates in a representative experiment. As shown in FIGS. 8A and 8B,the 5D5 Fab acts as an antagonist at concentrations as low as 1 μg/ml,significantly blocking BaF3-hmet cell proliferation in the presence ofHGF.

B. Human Mammary Tumor Cell Assay

Antagonistic activity of 5D5 Fab fragments was examined in a mitogenicassay to measure induction of DNA synthesis in a human breast carcinomacell line. MDA-MB-435 human breast carcinoma cells (ATCC HTB 129) (whichexpress c-Met) were cultured in DMEM, 5% fetal bovine serum, 100 U/mlpenicillin, 100 μg/ml streptomycin sulfate and 2 mM glutamine. Prior toconducting the assay, the cells were washed and resuspended in assaymedium (DMEM, 0.1% BSA, 2 mM glutamine). The cells were then plated in a96 well plate at 5,000 cells/well and incubated at 37° C. with varyingconcentrations of rhuHGF (0, 0.1, 1, 10, 100, 1000 ng/ml) in the absenceor presence of 10 μg/ml 5D5 Fab (Example 7) overnight. Next, 1 μCi³H-thymidine was added to each well and the plates were incubated for 24hours at 37° C. The cells were then harvested and the amount ofradioactivity incorporated into the DNA was quantitated in a microplatescintillation counter. The results are shown in FIG. 9. Data are themean±SEM of 6 replicates in a representative experiment. As shown inFIG. 9, the mitogenic response of the carcinoma cells to the HGF wascompletely blocked by 5D5 Fab.

Example 10 Effect of 5D5 Fab on Tyrosine Phosphorylation of c-Met

The ability of 5D5 Fab to stimulate the c-Met receptor or induce c-Metactivation was examined in an in vitro assay measuring c-Met tyrosinephosphorylation. Phosphorylation of c-Met was measured in a sandwichELISA, based on the methods of Sadick et al. in which solubilized c-Metis captured onto a plate coated with rabbit anti-c-Met polyclonal IgGand detected with anti-P-Tyr [Sadick et al., Anal. Biochem., 235:207-214(1996)]. Microtiter plates were coated overnight at 4° C. with 5 μg/mlrabbit anti-c-Met polyclonal IgG, then non-specific binding was blockedas described in Example 3. While the microtiter plates were coated, A549cells (described in Example 1) were plated into 100 mm dishes. The nextday the cells were washed twice with assay medium (MEM supplemented with1% BSA) and then challenged for 10 minutes with NK1 (1 μg/ml) [NK1prepared as described in Lokker et al., J. Biol. Chem., 268:17145-17150(1993)] or 5D5 Fab alone (10 μg/ml) (Example 7) or with rhuHGF (10ng/ml) (Example 3). The cells were washed twice with PBS, and then lysedin 1 ml lysis buffer (PBS, 0.2% Triton X-100, 10 μg/ml aprotonin, 5 mMNaF, 2 mM sodium orthovanadate, and 0.2 mM PMSF) for 30 minutes on anorbital shaker at room temperature. The lysate was centrifuged for 10minutes and 100 μl supernatant was transferred in duplicate to theblocked microtiter plates. After incubation for 2 hours at 23° C.,tyrosine phosphorylation was detected by incubation for 2 hours at 23°C. with biotin-anti-P-Tyr (Upstate Biotech, Lake Placid, N.Y.), followedby HRP-streptavidin. Next, TMB peroxidase substrate (KPL, Gaithersburg,Md.) was added. The reaction was stopped with phosphoric acid and OD wasmeasured at 450 nm in an automatic plate reader. Total c-Met wasmeasured in parallel wells incubated as above except that detection waswith biotinylated rabbit anti-c-Met polyclonal IgG (NIHS biotin, PierceChemical).

As shown in FIGS. 10A and 10B, lysates were analyzed for relativeamounts of tyrosine phosphorylated c-Met (FIG. 10A) and total c-Met(FIG. 10B). Results shown in FIGS. 10A and 10B are the mean±SEM of 4replicates from a representative experiment. NK1 did stimulate tyrosinephosphorylation of c-Met (indicating agonistic activity), but did notsignificantly inhibit the response of A549 cells to the HGF. Incontrast, 5D5 Fab had no stimulatory effect on tyrosine phosphorylationand blocked HGF responses.

Example 11 Effect of Heparin on Antagonistic Activity of 5D5 Fab

The effect of heparin on the antagonistic activity of 5D5 Fab wasexamined using a thymidine incorporation assay, as described in Examples6 and 9 except that BaF3-hmet cells were incubated with NK1 (describedin Example 10; 0, 0.01, 0.1, 1 μg/ml), 5D5 Fab (described in Example 7;0, 0.01, 0.1, 1, 10 μg/ml) or 5D5 Fab and 10 ng/ml HGF. Each incubationwas done in the absence or presence of heparin (1 μg/ml) (Sigma). Theresults, shown in FIGS. 11A-11C, are the mean±SEM of 4 replicates from arepresentative experiment.

As shown in FIG. 11A, NK1 can be converted to an agonistic molecule bythe presence of exogenous heparin. In contrast, heparin did not conferagonist activity on the 5D5 Fab (FIG. 11B). Although the response to HGFwas enhanced by heparin, the 5D5 Fab remained an antagonist andcompletely blocked HGF activity (FIG. 11C).

Example 12 Effect of 5D5 Fab on HGF-Induced Cell Migration

An assay was conducted to examine the ability of 5D5 Fab to inhibitHGF-induced cell migration. A549 cells (described in Example 1) wereadded to the upper wells of a 48-well modified Boyden chamber (NEUROPROBE INC., Cabin John, Md.) containing rhuHGF (10 ng/ml) and/or 5D5 Fab(Example 7; 10 μg/ml) in the lower wells. A barrier ofpolyvinylpyrrolidone-free polycarbonate filter with 8 micron pore sizewas employed. After incubation for 6 hours at 37° C., the cells on theupper surface of the membrane were scraped off, and the membrane wasstained with DifQuik™ (Baxter Scientific Products). A549 cells that hadmigrated onto the lower side of the membrane were counted in 20 randomlyselected fields for each well. The data shown in Table 1 below are themean±SD of 4 wells.

TABLE 1 Inhibition of HGF-induced migration by 5D5 Fab Number of CellsMigrated per High Powered Field Experiment 1 Experiment 2 Control 0 ± 09 ± 8 HGF 224 ± 30  35 ± 10 5D5 Fab 2 ± 2 7 ± 4 HGF + 5D5 Fab 37 ± 22 6± 4

The results show that 5D5 Fab blocked migration responses to HGF.

Example 13 Sequencing, Cloning and Expression of 5D5 Fab

An aliquot of 5D5 Fab (Example 7) was resolved on a 4-20% gradient SDSgel and electroblotted onto a PVDF (Immobilon-PSQ) membrane (Millipore,Marlborough, Mass.) for 1 hour at 250 mA constant current in a BioRadTrans-Blot transfer cell [Matsudaira, J. Biol. Chem., 262:10035-10038(1987)]. The membrane was then stained with 0.1% Coomassie Blue R-250 in50% methanol for 30 seconds and destained for 2-3 minutes with 10%acetic acid in 50% methanol. After destaining, the membrane wasthoroughly washed with water and allowed to dry before sequencing on amodel 473A automated protein sequencer, using a Blott™ cartridge(Applied Biosystems). Peaks were integrated with Justice Innovationsoftware using Nelson Analytical 760 interfaces and sequenceinterpretation was performed on a DEC alpha [Henzel et al., J.Chromatography, 404:41-52 (1996)].

Obtaining sequence of the 5D5 heavy chain required deblocking, which wasperformed as follows. The Fab fragment was reduced with 7 mM DTT at 45°C. for 1 hour and alkylated with 180 mM isopropylacetamide at 25° C. for20 minutes [Krutzsch et al., Anal. Biochem., 209:109-116 (1993)]. Thealkylated Fab fragment was then exchanged 3× in a Microcon-10 with 0.1 Msodium phosphate containing 10 mM DTT (in digestion buffer) and digestedwith 1 mU of pyroglumate aminopeptidase (Takara Biochemicals, Berkeley,Calif.) at 45° C. for 3 hours in 20 μl digestion buffer. The deblockedFab was then transferred to the PVDF membrane and sequenced as describedabove.

N-terminal sequence data were used to design PCR primers specific forthe 5′ ends of the variable regions of the light and heavy chains, while3′ primers were designed to anneal to the concensus framework 4 of eachchain [Kabat et al., Sequences of Proteins of Immunological Interest,Public Health Service, National Institutes of Health, Bethesda, Md.,(1991)]. The primers were also designed to add restriction enzyme sitesfor cloning. Total RNA, extracted from 10⁸ cells of hybridoma 5D5 with aStratagen RNA isolation kit, was used as substrate for RT-PCR. Reversetranscription was performed under standard conditions [Kawasaki et al.,PCR Protocols: A Guide to Methods and Applications, Innis et al., eds.,Academic Press, San Diego, pp. 21-27 (1990)] using the framework 4specific primers and Superscript II RNase H-Reverse Transcriptase(Gibco-BRL, Gaithersburg, Md.). PCR amplification employed Taqpolymerase (Perkin Elmer-Cetus, Foster City, Calif.), as described inKawasaki et al., supra, except that 2% DMSO was included in the reactionmixture. Amplified DNA fragments were digested with restriction enzymesSfiI and RsrII (light chain) or MluI and ApaI (heavy chain), gelpurified, and cloned into a derivative of expression plasmid pAK19[Carter et al., Bio/Technology, 10:163-167 (1992)]. This vector,pXCA730, was modified by site-directed mutagenesis [Kunkel et al., Proc.Natl. Acad. Sci., 82:488 (1985)] to contain unique restriction sitesbetween the STII signal sequences and the variable domains, and at thejunction of the variable and constant domains of each chain. The lightand heavy chain variable domain cDNAs were inserted upstream and inframe to human CK and CH1 domains. The C-terminal cysteine of the heavychain in pAK19, which could form a disulfide bridge to give F(ab′)₂molecules, was removed to permit expression of only the Fab form of theantibody.

Recombinant 5D5 Fab was expressed in E. coli K12 strain 33B6 [Rodrigueset al., Cancer Res., 55:63-70 (1995)], as described by Carter et al.,supra. FIG. 12 shows a schematic representation of plasmid p5D5containing the discistronic operon for expression of the chimer 5D5 Fab.Expression was under the control of the E. coli alkaline phosphatasepromoter, which is inducible by phosphate starvation. Each antibodychain was preceded by the E. coli heat-stable enterotoxin II signalsequence to direct sequence to the periplasmic space of E. coli. Themurine variable domains from antibody 5D5 (V_(L) and V_(H)) wereprecisely fused on their 3′ side to human K₁ C_(L) and C_(H1) constantdomains, respectively.

The cell pellet from a 10-L fermentation was harvested by continuousfeed centrifugation, frozen and stored at −70° C. A portion of thepellet was suspended in extraction buffer (120 mM MES, pH 6.0, and 5 mMEDTA, 5 ml/gram of paste). The suspension was mixed thoroughly using anultraturrax (Janke and Kunkel) for approximately 15 minutes at 4° C.Intact cells were then disrupted using 2 passes through a cellhomogenizer (Microfluidizer, by Microfluidics Corp., Newton, Mass.)fitted with a cooling coil. The suspension was then adjusted to 0.1%(v/v) polyethyleneimine using a 5% (v/v) stock which had been adjustedto pH 6.0. Intact cells and PEI-flocculated debris were separated fromthe soluble fraction by centrifugation at 25,400×g for 30 minutes. Thesupernatant was adjusted to a conductivity less than 4 mS by addition ofpurified water and loaded onto a column (1×10 cm) of Bakerbond ABX, 40micron particle size (J. T. Baker, Phillipsburg, N.J.). The column hadbeen equilibrated in 50 mM MES, 5 mM EDTA, pH 6.0. All steps were doneat a linear flow rate of 100 cm/hour. After loading, the column waswashed with equilibration buffer until the absorbance of the columneffluent was equivalent to baseline. Elution was performed using a16-column volume, linear gradient from 0 to 100 mM ammonium sulfate inequilibration buffer. Column fractions were analyzed bySDS-polyacrylamide gel electrophoresis and fractions which contained theFab were pooled. The conductivity of the pool from the ABX column waslowered to less than 4 mS and loaded onto a column (1×10 cm) ofSP-Sepharose High Performance resin (Pharmacia Biotech, Piscataway,N.J.) that had been equilibrated in 25 mM MOPS buffer, pH 6.9. All stepswere performed at a linear flow rate of 100 cm/hour. Following the load,the column was washed with one column volume of equilibration buffer.The 5D5 Fab was then eluted from the column using a 16-column volume,linear gradient from 0 to 200 mM sodium acetate in equilibration buffer.Column fractions were analyzed by SDS-polyacrylamide gel electrophoresisand fractions which contained the Fab were pooled.

The light chain of the 5D5 Fab included amino acid residues 1 to 220, asshown in FIG. 1A (SEQ ID NO:1), and the heavy chain included amino acidresidues 1 to 230 (wherein amino acid residue 1 comprised a glutamineacid residue), as shown in FIG. 1B (SEQ ID NO:2). Molecular weightanalysis of the 5D5 Fab showed that it had a molecular weight ofapproximately 45 kDa. Although not fully understood, it is believed thatamino acid residue 1 of native 5D5 Fab heavy chain may be a glutamineresidue.

Example 14 Assay of Recombinant 5D5 Fab Binding to c-Met

An inhibition assay was conducted, essentially as described in Examples3 and 8, to examine the ability of recombinant 5D5 Fab (Example 13) toblock binding of HGF to c-Met-IgG fusion protein. rhuHGF, recombinant5D5 Fab, or a recombinant control Fab (anti-VEGF Fab, Genentech, Inc.)were tested at concentrations ranging from 0.001-10 μg/ml, as shown inFIG. 13. The data, shown in FIG. 13, is the mean±SD of duplicate wells.The graph in FIG. 13 illustrates that the recombinant 5D5 Fab inhibitedHGF binding to c-Met while the control did not.

Example 15 Effect of Heparin on Antagonistic Activity of Recombinant 5D5Fab

The effect of heparin on antagonistic activity of recombinant 5D5 Fab(Example 13) was examined using a thymidine incorporation assay asdescribed in Examples 6, 9 and 11. BaF3-hmet cells were incubated with0-10,000 ng/ml recombinant 5D5 Fab (Example 13) or recombinant controlFab alone (anti-VEGF Fab; described in Example 14), with 1 μg/ml heparin(Sigma), with 10 ng/ml rhuHGF, or with 10 ng/ml rhuHGF plus 1 μg/mlheparin.

The results are shown in FIGS. 14A-14D, and the data are the mean±SEM of4 replicates in a representative experiment. The results show that therecombinant 5D5 Fab remained an antagonist in the presence and absenceof heparin.

Deposit of Materials

The following cultures have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20220-2209USA (ATCC):

Hybridoma ATCC No. Deposit Date 1A3.3.13 HB-11894 May 23, 1995 5D5.11.6HB-11895 May 23, 1995

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of deposit. The organisms will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the cultures to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrains are not to be construed as a license to practice the inventionin contravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cultures deposited, sincethe deposited embodiments are intended as an illustration of an aspectof the invention and any cultures that are functionally equivalent arewithin the scope of this invention. The deposit of material herein doesnot constitute an admission that the written description hereincontained is inadequate to enable the practice of any aspect of theinvention, including the best mode thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustration that itrepresents.

1. A hepatocyte growth factor (HGF) receptor antagonist whichspecifically binds to a HGF receptor.
 2. The antagonist of claim 1 whichis an antibody.
 3. The antibody of claim 2 which is a monoclonalantibody.
 4. The antibody of claim 2 wherein the antibody binds to thec-Met receptor.
 5. The antibody of claim 4 wherein the antibody inhibitsbinding of human HGF to the c-Met receptor.
 6. The antibody of claim 2which comprises a Fab fragment.
 7. The antibody of claim 2 comprising achimeric antibody.
 8. The antibody of claim 2 having the biologicalcharacteristics of the monoclonal antibody produced by the hybridomacell line deposited under American Type Culture Collection AccessionNumber ATCC HB-11894.
 9. The antibody of claim 2 wherein the antibodybinds to substantially the same epitope as the epitope to which themonoclonal antibody produced by the hybridoma cell line deposited underAmerican Type Culture Collection Accession Number ATCC HB-11894 binds.10. The antibody of claim 2 having the biological characteristics of themonoclonal antibody produced by the hybridoma cell line deposited underAmerican Type Culture Collection Accession Number ATCC HB-11895.
 11. Theantibody of claim 2 wherein the antibody binds to substantially the sameepitope as the epitope to which the monoclonal antibody produced by thehybridoma cell line deposited under American Type Culture CollectionAccession Number ATCC HB-11895 binds.
 12. An isolated HGF receptorantagonist which specifically binds to a HGF receptor and comprisesamino acid residues 1-220 of FIG. 1A and amino acid residues 1-230 ofFIG. 1B.
 13. An isolated nucleic acid encoding the HGF receptorantagonist of claim
 12. 14. An isolated nucleic acid encoding the HGFreceptor antagonist of claim
 1. 15. The nucleic acid of claim 14 whereinsaid antagonist is an antibody.
 16. A vector comprising the nucleic acidof claim
 12. 17. A host cell comprising the vector of claim
 16. 18. Amethod of producing HGF receptor antagonist comprising culturing thehost cell of claim 17 and recovering the HGF receptor antagonist fromthe host cell culture.
 19. A hybridoma cell line which produces theantibody of claim
 3. 20. The hybridoma of claim 19 comprising ATCCHB-11894.
 21. The hybridoma of claim 19 comprising ATCC HB-11895.
 22. Achimeric molecule comprising the HGF receptor antagonist of claim 1 orclaim 12 fused to a heterologous polypeptide sequence.
 23. The chimericmolecule of claim 22 wherein said heterologous polypeptide is a tagpolypeptide sequence.
 24. The chimeric molecule of claim 22 wherein saidheterologous polypeptide sequence is an immunoglobulin sequence.
 25. Thechimeric molecule of claim 22 wherein said heterologous polypeptidesequence is an albumin sequence.
 26. A pharmaceutical compositioncomprising the HGF receptor antagonist of claim 1 of claim 12 and apharmaceutically-acceptable carrier.
 27. The pharmaceutical compositionof claim 26 wherein said antagonist is an antibody.
 28. A method oftreating cancer in a mammal, comprising administering an effectiveamount of HGF receptor antagonist to a mammal diagnosed as havingcancer.
 29. The method of claim 28 wherein said antagonist is anantibody.
 30. The method of claim 28 wherein said cancer is breastcancer.
 31. The method of claim 28 wherein said cancer is pancreaticcancer.
 32. The method of claim 28 wherein said cancer is colon cancer.33. The method of claim 28 wherein said cancer is lung cancer.
 34. Anarticle of manufacture, comprising: a container; a label on saidcontainer; and a composition contained within said container; whereinthe composition includes an active agent effective for treating cancer,the label on said container indicates that the composition can be usedfor treating cancer, and the active agent in said composition comprisesHGF receptor antagonist.
 35. The article of manufacture of claim 34wherein said antagonist is an antibody.
 36. The article of manufactureof claim 34 further comprising instructions for administering the HGFreceptor antagonist to a mammal.
 37. A kit, comprising: a firstcontainer, a label on said container, and a composition contained withinsaid container; wherein the composition includes an active agenteffective for treating cancer, the label on said container indicatesthat the composition can be used for treating cancer, and the activeagent in said composition comprises HGF receptor antagonist; a secondcontainer comprising a pharmaceutically-acceptable buffer; andinstructions for using the HGF receptor antagonist to treat cancer. 38.An article of manufacture, comprising: a container; a label on saidcontainer; and a composition contained within said container; whereinthe composition includes an active agent effective for detecting orpurifying HGF receptor, the label on said container indicates that thecomposition can be used for detecting or purifying HGF receptor, and theactive agent in said composition comprises HGF receptor antagonist. 39.The article of manufacture of claim 38 wherein said antagonist is anantibody.
 40. A kit, comprising: a first container, a label on saidcontainer, and a composition contained within said container; whereinthe composition includes an active agent effective for detecting orpurifying HGF receptor, the label on said container indicates that thecomposition can be used for detecting or purifying HGF receptor, and theactive agent in said composition comprises HGF receptor antagonist; asecond container comprising a pharmaceutically-acceptable buffer; andinstructions for using the HGF receptor antagonist to detect or purifyHGF receptor.